Anti-BRG1 antibody [EPNCIR111A]

23 product images

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  Western blot - Anti-BRG1 antibody [EPNCIR111A] (ab110641)

  False colour image of Western blot: Anti-BRG1 antibody [EPNCIR111A] staining at 1/1000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab110641 was shown to bind specifically to BRG1. A band was observed at 185 kDa in wild-type HEK-293T cell lysates with no signal observed at this size in SMARCA4 knockout cell line Human SMARCA4 (BRG1) knockout HEK-293T cell line ab255432 (knockout cell lysate Human SMARCA4 (BRG1) knockout HEK-293T cell lysate ab263853). To generate this image, wild-type and SMARCA4 knockout HEK-293T cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) at 1/20000 dilution.

  All lanes: Western blot - Anti-BRG1 antibody [EPNCIR111A] (ab110641) at 1/1000 dilution

  Lane 1: Wild-type HEK-293T cell lysate at 20 µg

  Lane 2: SMARCA4 knockout HEK-293T cell lysate at 20 µg

  Lane 2: Western blot - Human SMARCA4 (BRG1) knockout HEK-293T cell line (Human SMARCA4 (BRG1) knockout HEK-293T cell line ab255432)

  Performed under reducing conditions.

  Predicted band size: 185 kDa

  Observed band size: 185 kDa

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  Western blot - Anti-BRG1 antibody [EPNCIR111A] (ab110641)

  Lanes 1- 2: Merged signal (red and green). Green - ab110641 observed at 185 kDa. Red - Anti-alpha Tubulin antibody [DM1A] - Loading Control (Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291) observed at 50 kDa.
  

  ab110641 was shown to react with SMARCA4 in wild-type HEK-293T cells in western blot. Loss of signal was observed when knockout cell line Human SMARCA4 (BRG1) knockout HEK-293T cell line ab255432 (knockout cell lysate Human SMARCA4 (BRG1) knockout HEK-293T cell lysate ab263853) was used. Wild-type HEK-293T and SMARCA4 knockout HEK-293T cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab110641 and Anti-alpha Tubulin antibody [DM1A] - Loading Control (Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291) overnight at 4°C at a 1 in 10000 dilution and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^®800CW) preadsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye^®680RD) preadsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

  All lanes: Western blot - Anti-BRG1 antibody [EPNCIR111A] (ab110641) at 1/10000 dilution

  Lane 1: Wild-type HEK-293T cell lysate at 20 µg

  Lane 2: SMARCA4 knockout HEK-293T cell lysate at 20 µg

  Lane 2: Western blot - Human SMARCA4 (BRG1) knockout HEK-293T cell line (Human SMARCA4 (BRG1) knockout HEK-293T cell line ab255432)

  Performed under reducing conditions.

  Predicted band size: 185 kDa

  Observed band size: 185 kDa

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  Immunoprecipitation - Anti-BRG1 antibody [EPNCIR111A] (ab110641)

  BRG1 was immunoprecipitated from 0.35 mg HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate 10μg with ab110641 at 1:20 dilution (0.3μg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab110641 1:1000 dilution (0.12 μg/ml). VeriBlot for IP Detection Reagent (HRP) (VeriBlot for IP Detection Reagent (HRP) ab131366) was used as the secondary antibody at 1:1000 dilution.
  

  Lane 1: HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate 10μg

  Lane 2: ab110641 IP in HeLa whole cell lysate

  Lane 3: Rabbit monoclonal IgG (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) instead of ab110641 in HeLa whole cell lysate.

  Blocking and dilution buffer and concentration: 5% NFDM/TBST.

  Exposure time: 1 second

  All lanes: Immunoprecipitation - Anti-BRG1 antibody [EPNCIR111A] (ab110641)

  Predicted band size: 185 kDa

  Observed band size: 185 kDa

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  Immunocytochemistry/ Immunofluorescence - Anti-BRG1 antibody [EPNCIR111A] (ab110641)

  BRG1 immunofluorescence staining of HeLa cells using rabbit anti-BRG1 antibody
  
  ab110641 staining BRG1 in HeLa cells. The cells were fixed with 4% formaldehyde (10min) permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab110641 at 1/500 dilution and Alexa Fluor® 594 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker ab195889 (Mouse monoclonal [DM1A] to alpha Tubulin - Microtubule Marker (Alexa Fluor^® 594)) at 1/250 dilution overnight at +4°C followed by a further incubation at room temperature for 1h with Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081 (Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor^® 488)) at 2 μg/ml (shown in green). Nuclear DNA was labelled in blue with DAPI.

  Image was taken with a confocal microscope (Leica-Microsystems TCS SP8).

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-BRG1 antibody [EPNCIR111A] (ab110641)

  Immunohistochemical analysis of paraffin-embedded Human cervical carcinoma tissue labeling BRG1 with ab110641, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Nuclear staining on human cervical carcinoma. Counterstained with Hematoxylin. Heat mediated antigen retrieval using Antigen Retrieval Buffer (100X Tris-EDTA Buffer, pH 9.0) ab93684 (Tris/EDTA buffer, pH 9.0).
  

  Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).

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  Western blot - Anti-BRG1 antibody [EPNCIR111A] (ab110641)

  Blocking/Diluting buffer and concentration: 5% NFDM/TBST.
  

  Exposure time: Lane 1 to 4: 5 seconds; Lane 5: 15 seconds

  All lanes: Western blot - Anti-BRG1 antibody [EPNCIR111A] (ab110641) at 1/10000 dilution

  Lane 1: HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysates at 20 µg

  Lane 2: NIH/3T3 (Mouse embryonic fibroblast) whole cell lysates at 20 µg

  Lane 3: RAW 264.7(Mouse Abelson murine leukemia virus-induced tumor macrophage)whole cell lysates at 20 µg

  Lane 4: C6 (Rat glial tumor glial cell) whole cell lysates at 20 µg

  Lane 5: K-562 (Human chronic myelogenous leukemia lymphoblast) whole cell lysates at 20 µg

  Secondary

  All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/20000 dilution

  Predicted band size: 110 kDa, 185 kDa

  Observed band size: 17 kDa, 185 kDa, 36 kDa

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  Western blot - Anti-BRG1 antibody [EPNCIR111A] (ab110641)

  Lanes 1 - 4: Merged signal (red and green). Green - ab110641 observed at 185 kDa. Red - loading control, ab18058, observed at 124 kDa.
  

  ab110641 was shown to specifically react with BRG1 in wild-type HAP1 cells. No band was observed when BRG1 knockout samples were used. Wild-type and BRG1 knockout samples were subjected to SDS-PAGE, ab110641 and ab18058 (loading control to Vinculin) were both diluted 1/10,000 and incubated overnight at 4°C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) secondary antibodies at 1/10,000 dilution for 1hr at room temperature before imaging.

  All lanes: Western blot - Anti-BRG1 antibody [EPNCIR111A] (ab110641) at 1/10000 dilution

  Lane 1: Wild-type HAP1 cell lysate at 20 µg

  Lane 2: BRG1 knockout HAP1 cell lysate at 20 µg

  Lane 3: K562 cell lysate at 20 µg

  Lane 4: Molt-4 cell lysate at 20 µg

  Predicted band size: 185 kDa, 36 kDa

  Observed band size: 185 kDa

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  Flow Cytometry (Intracellular) - Anti-BRG1 antibody [EPNCIR111A] (ab110641)

  BRG1 flow cytometry staining of HeLa cells using rabbit anti-BRG1 antibody
  
  Intracellular Flow Cytometry analysis of HeLa (human cervix adenocarcinoma) cells labeling BRG1 with purified ab110641 at 1/200 dilution (10μg/ml) (red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. A Goat anti rabbit IgG (Alexa Fluorr^® 488) (1/2000 dilution) was used as the secondary antibody. Rabbit monoclonal IgG (Black) was used as the isotype control cells without incubation with primary antibody and secondary antibody (Blue) were used as the unlabeled control.

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  Immunocytochemistry/ Immunofluorescence - Anti-BRG1 antibody [EPNCIR111A] (ab110641)

  ab110641 staining BRG1 in wild-type HAP1 cells (top panel) and BRG1 knockout HAP1 cells (bottom panel). The cells were fixed with 4% formaldehyde (10min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab110641 at 1/500 dilution and Alexa Fluor® 594 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker ab195889 at 1/250 dilution (shown in pseudocolour red) overnight at +4°C, followed by a further incubation at room temperature for 1h with a goat secondary antibody to Rabbit IgG (Alexa Fluor® 488) (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081) at 2 μg/ml (shown in green). Nuclear DNA was labelled in blue with DAPI.
  

  Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

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  Western blot - Anti-BRG1 antibody [EPNCIR111A] (ab110641)

  Different batches of ab110641 were tested on HeLa (Human cervix adenocarcinoma epithelial cell) lysate at 0.5 µg/ml. 15 µg of lysate was loaded in each lane. Bands observed at 185 kDa.

  All lanes: Western blot - Anti-BRG1 antibody [EPNCIR111A] (ab110641)

  Predicted band size: 185 kDa

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-BRG1 antibody [EPNCIR111A] (ab110641)

  BRG1 immunohistochemistry staining of rat liver using rabbit anti-BRG1 antibody
  
  Immunohistochemical analysis of paraffin-embedded Rat liver tissue labeling BRG1 with ab110641, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Nuclear staining on rat liver. Counterstained with Hematoxylin. Heat mediated antigen retrieval using Antigen Retrieval Buffer (100X Tris-EDTA Buffer, pH 9.0) ab93684 (Tris/EDTA buffer, pH 9.0). Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-BRG1 antibody [EPNCIR111A] (ab110641)

  BRG1 immunohistochemistry staining of mouse kidney using rabbit anti-BRG1 antibody
  
  Immunohistochemical analysis of paraffin-embedded Mouse kidney tissue labeling BRG1 with ab110641, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Nuclear staining on mouse kidney. Counterstained with Hematoxylin. Heat mediated antigen retrieval using Antigen Retrieval Buffer (100X Tris-EDTA Buffer, pH 9.0) ab93684 (Tris/EDTA buffer, pH 9.0).
  

  Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-BRG1 antibody [EPNCIR111A] (ab110641)

  BRG1 immunohistochemistry staining of human kidney using rabbit anti-BRG1 antibody
  
  Immunohistochemical analysis of paraffin-embedded human kidney tissue staining BRG1 with ab110641 at 1/100 dilution. Heat mediated antigen retrieval was perfomed with citrate buffer (pH 6).

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-BRG1 antibody [EPNCIR111A] (ab110641)

  BRG1 immunohistochemistry staining of human testis using rabbit anti-BRG1 antibody
  
  Immunohistochemical analysis of paraffin-embedded human testis tissue staining BRG1 with ab110641 at 1/100 dilution. Heat mediated antigen retrieval was perfomed with citrate buffer (pH 6).

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  Flow Cytometry (Intracellular) - Anti-BRG1 antibody [EPNCIR111A] (ab110641)

  Flow cytometry overlay histogram showing left wild-type Hap1 positive cells and right negative SMARCA4 knockout Hap1 stained with Anti-Glypican 3 antibody [SP86] ab95363 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilised with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS containing 10% normal goat serum to block non-specific protein-protein interaction followed by the antibody (ab110641) (1x 106 in 100μl at 0.008 μg/ml (1/266250)) for 30min at 22°C.
  
  The secondary antibody Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed was incubated at 1/4000 for 30min at 22°C
  
  Isotype control antibody Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control (black line) was used at the same concentration and conditions as the primary antibody. Unlabelled sample was also used as a control (blue line).
  
  Acquisition of >5000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter.

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-BRG1 antibody [EPNCIR111A] (ab110641)

  BRG1 immunohistochemistry staining of human tonsil using rabbit anti-BRG1 antibody
  
  Immunohistochemical analysis of formalin fixed paraffin embedded human tonsil labelling BRG1 with ab110641 at a concentration of 0.1 µg/ml. The immunostaining was performed on a Ventana DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument with an OptiView DAB IHC Detection Kit. Heat mediated antigen retrieval was conducted for 32 mins at 100°C with ULTRA cell conditioning solution (CC1, pH 8.5). ab110641 anti-BRG1 antibody [EPNCIR111A] was incubated at 37°C for 16 mins. Sections were counterstained with Hematoxylin II. Image inset shows absence of staining in secondary antibody only control.

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-BRG1 antibody [EPNCIR111A] (ab110641)

  BRG1 immunohistochemistry staining of human tonsil using rabbit anti-BRG1 antibody
  
  Immunohistochemical analysis of formalin fixed paraffin embedded human tonsil labelling BRG1 with ab110641 at a concentration of 0.1 µg/ml. The immunostaining was performed on a Leica Biosystems BOND® RX instrument with a Bond™ Polymer Refine Detection kit. Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution 2) for 20 mins. ab110641 anti-BRG1 antibody [EPNCIR111A] was incubated for 30 mins at room temperature. Sections were counterstained with Hematoxylin. Image inset shows absence of staining in secondary antibody only control.

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  ChIC/CUT&RUN sequencing - Anti-BRG1 antibody [EPNCIR111A] (ab110641)

  ChIC/CUT&RUN was performed using a pAG-MNAse at a final concentration of 700 ng/mL, 2.5 x 10^5 HeLa (Human cervix adenocarcinoma epithelial cell line) cells and 5µg of ab110641 [EPNCIR111A]. The resulting DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 10 million reads. The negative IgG control Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730 is also shown.
  
  Additional screenshots of mapped reads can be found in the Protocol booklet in the Support and downloads section.
  
  The University of Geneva owns patents relevant to ChIC (Chromatin Immuno-Cleavage) methods.

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  Western blot - Anti-BRG1 antibody [EPNCIR111A] (ab110641)

  Western Blotting using Anti-BRG1 antibody [EPNCIR111A], ab110641. Publication image from Liu, M. et al., 2019, Nat Commun, 31601814. Legend direct from paper.

  Brg1 loss in adult intestines leads to the development of colitis. Two-month-old mice (Brg1flox/flox or VillinCre-ERT2; Brg1flox/flox) were treated with tamoxifen, and thereby generated Brg1F/F or Brg1IEC-AKO mice. a Immunohistochemical (upper panel) and immunoblot (IB) analyses (lower panel) of BRG1 expression are shown. b Representative histological images of middle-distal sections of Brg1F/F and Brg1IEC-AKO mice are shown at the indicated time points, and semi-quantitative scoring of the histopathology is shown (n = 12 per genotype). Arrow: immune cell infiltration. c Colonic lamina propria cells isolated from 16-week-old mice (2 months after the Brg1 ablation) are analyzed by flow cytometry (n = 4 per genotype). d RT-qPCR analysis of colon homogenates from 16-week-old mice to assess cytokine and chemokine productions (n = 6 per genotype). e Alcian blue-Periodic acid Schiff (AB-PAS; goblet cells) staining and lysozyme (Lys; Paneth cells) staining of the intestines derived from 3-month-old mice (1 month after the Brg1 ablation) and quantitation results are shown in the right (n = 5). f Survival rate of Brg1IEC-AKO mice compared with that of Brg1F/F mice after the 3% DSS treatment (n = 10). g–i Mice were fed 1% DSS in their drinking water and loss of body weights (g) and colon length (h) are recorded (n = 10 per genotype). i H&E-stained sections of middle-distal colon tissue collected on day 10 from 1% DSS-treated mice, and the quantitation of histology score are shown in the right. The data represent the mean ± S.E.M., and statistical significance was determined by a two-tailed Student’s t-test. *p < 0.05, **p < 0.01, and ***p < 0.01. Scale bars: 50 μm (a), 100 μm (b, e, i), 1 cm (h). N.S., not significant, SI: small intestine.

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  Image collected and cropped by CiteAb under a CC-BY license from the publication

  Immunohistochemistry - Anti-BRG1 antibody [EPNCIR111A] (ab110641)

  Immunohistochemistry using Anti-BRG1 antibody [EPNCIR111A], ab110641. Publication image from Liu, M. et al., 2019, Nat Commun, 31601814. Legend direct from paper.

  BRG1 expression is decreased in IBD patients. a Box plot of BRG1 mRNA in healthy controls and IBD specimens (using dataset GSE9452 and GSE3365). In boxplots (middle line depicts the median and the whiskers the min-to-max range). b RT-qPCR analysis of BRG1 mRNA in IBD specimens and healthy subjects (n = 81 per group). c BRG1 staining images are shown in the upper panel, and epithelial BRG1 expressions in normal and IBD biopsies is quantified in the bottom panel (χ2 test). Staining indexes use a 10-point quantification scale, and a score > 4 is considered higher level. Scale bar: c 50 μm. The data represent the mean ± S.E.M., and statistical significance was determined by a two-tailed Student’s t-test unless otherwise indicated. ***p < 0.001.

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  Image collected and cropped by CiteAb under a CC-BY license from the publication

  Western blot - Anti-BRG1 antibody [EPNCIR111A] (ab110641)

  Western Blotting using Anti-BRG1 antibody [EPNCIR111A], ab110641. Publication image from Liu, M. et al., 2019, Nat Commun, 31601814. Legend direct from paper.

  BRG1 overexpression in IECs protects the mice from colitis and tumorigenesis. a Scheme of R26Brg1/+ mice and conditional overexpression of BRG1 in IECs (Brg1IEC-OE/+) mice. b Immunohistochemistry and western blotting analysis of BRG1 expression in colonic tissues of R26Brg1/+ and Brg1IEC-OE/+ mice. c, d DSS was administered for 5 days, and the colon lengths (c) and body weights (d) are recorded (n = 10 per genotype). e Representative H&E-stained middle-distal colon sections and histology score (right) from R26Brg1/+ and Brg1IEC-OE/+ mice treated with 3% DSS (n = 10 per genotype). The arrow indicates immune cell infiltration. f RT-qPCR analysis of the relative mRNA levels of the indicated genes in whole colonic homogenates from untreated or DSS-treated mice (day 5, n = 4 per genotype). g Alcian blue-Periodic acid Schiff (AB-PAS; goblet cells) staining and lysozyme (Lys; Paneth cells) staining of the intestines derived from 3% DSS-treated mice and quantitation results are shown in the right (n = 5). The 2-month-old mice were treated with AOM, followed by three cycles of 3% DSS treatment. h Macroscopic images of the mice after 3 months of AOM treatment, and the numbers the tumors based on the sizes are quantified in the right panel (n = 10 per genotype). i Representative images of H&E-stained colon sections as indicated; the percent grading of tumors is shown in the right (n = 10, χ2 test). The data represent the mean ± S.E.M., and statistical significance was determined by a two-tailed Student’s t-test unless otherwise indicated. *p < 0.05, **p < 0.01, and ***p < 0.01. Scale bars: 50 μm (b, g), 200 μm (e, bottom i), 1 cm (c, h, upper i).

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  Image collected and cropped by CiteAb under a CC-BY license from the publication

  Western blot - Anti-BRG1 antibody [EPNCIR111A] (ab110641)

  Western Blotting using Anti-BRG1 antibody [EPNCIR111A], ab110641. Publication image from Fernando, T. M. et al., 2020, Nat Commun, 33144586. Legend direct from paper.

  Differential effects of SMARCA4 mutants to rescue cell growth and chromatin accessibility loss after SMARCA2 knockdown.a Long term clonogenic growth of NCI-H1944 cells transduced with SMARCA4 WT or mutants after SMARCA2 knockdown. Representative of at least 3 replicates. b Immunoblot of cells from a (representative of at least 3 replicates). c Heatmap of ATAC-seq changes at sites after SMARCA2 knockdown in cells from a (n = 2 per construct). Values represent log2 fold-change relative to LACZ control after SMARCA2 knockdown. d Heatmap of SMARCA2 and SMARCA4 occupancy at regions with lower accessibility after SMARCA2 knockdown (sites from c) (n = 2 per construct). SMARCA2 ChIP-seq was performed in NCI-H1944 cells expressing LACZ. SMARCA4 ChIP-seq was performed in NCI-H1944 cells expressing SMARCA4 WT and doxycycline (DOX)-inducible expression of SMARCA2-targeting shRNA. Data are shown as normalized peak counts per million genomic DNA fragments in a 2 kb window around the peak center. Rows are rank ordered by SMARCA2 enrichment. R, replicate; INP, input. e Number of sites closed (left axis, blue bar, n = 2 per construct) and mean percent cell death (right axis, red dot, mean of 3 replicates) after SMARCA2 knockdown in cells from a. f Heatmap of genes downregulated after SMARCA2 knockdown in NCI-H1944 cells transduced with LACZ, WT or K785R mutant (n = 3 per construct). Data are shown as mean-centered normalized reads per kb of transcript per million mapped reads (nRPKM). g Long term clonogenic growth of CAL-12T, NCI-H1435 and HCC1897, which all harbor homozygous SMARCA4 missense mutations, after knockdown of SMARCA2 or SMARCA4 (left). Immunoblot confirming SMARCA2/SMARCA4 protein depletion (right). Data are representative of at least 2 replicates.

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  Image collected and cropped by CiteAb under a CC-BY license from the publication

  Western blot - Anti-BRG1 antibody [EPNCIR111A] (ab110641)

  Western Blotting using Anti-BRG1 antibody [EPNCIR111A], ab110641. Publication image from Imhof, A. et al., 2020, Nat Commun, 31964861. Legend direct from paper.

  Identification of PU.1 proximal proteins using BioID.a Schematic of the experimental setup. b Volcano plot illustrating proteins significantly enriched in PU.1-BirA-mRNA-transfected CTV-1 cells compared with NLS-BirA-mRNA-transfected control cells. Blue dots represent proteins with FDR < 0.05 and log fold change (logFC) > 2. Only PU.1-specific proteins of GO terms for chromatin organization and interesting transcriptional regulators are highlighted. c STRING analysis illustrating the functional protein association network. The network view summarizes predicted associations for proteins significantly enriched in the PU.1-BioID. The network nodes represent the proteins, the edges represent predicted functional associations. Only connected nodes are shown. d Dot plots showing the enrichment of peptides (ratios of log2-transformed normalized iBAQ values) representing the indicated SWI/SNF components (PU.1/SPI1 is shown as a control) in BioIDs from PU.1-BirA vs. NLS-BirA (control), ΔQ-BirA, or ΔA-BirA-mRNA-transfected CTV-1 cells (***P < 0.001; **P < 0.01; *P < 0.05; paired t-test, permutation-based correction). e Immunoblotting of αFLAG mAb immunoprecipitations (5 h after electroporation, IP 5 h), along with corresponding input lysates of control (mock), PU.1, ΔQ, ΔA, and BirA-mRNA-transfected cells using the indicated antibodies. f IGV genome browser tracks for the GSN locus showing SMARCA4 (BRG1) and PU.1 ChIP-seq, as well as ATAC-seq coverage in control (mutPU.1, gray–green)-, PU.1 (blue)-, ΔQ mutant (green)-, and ΔA mutant (lightbrown)-expressing cells. g Distribution of the PU.1 ChIP-seq, ATAC-seq signal, and SMARCA4 (BRG1) ChIP-seq signals in control (mutPU.1), PU.1, ΔQ, and ΔA mRNA-transfected cells across the 45 K clustered PU.1-binding sites, as well as the disappearing ~3 K sites that were accessible prior to PU.1 induction.