Anti-mCherry antibody

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-mCherry antibody (AB167453)

Immunofluorescent analysis of HEK293 cells transfected with pFin- EF1-mCherry vector labeling mCherry with ab167453 at 1/500 (red). Green antibody staining is only seen in cells which express mCherry, as expected, and the superimposition of the green and red signals results in an orange signal. Blue DNA stain.

• WB

Unknown

Western blot - Anti-mCherry antibody (AB167453)

All lanes:

Western blot - Anti-mCherry antibody (ab167453) at 1/1000 dilution

Lane 1:

HEK293 cells transfected with pFin- EF1-mCherry vector

Lane 2:

Non-transfected HEK293 cells

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• ICC/IF

CiteAb

Immunocytochemistry/ Immunofluorescence - Anti-mCherry antibody (AB167453)

mCherry Immunocytochemistry/ Immunofluorescence using Anti-mCherry antibody ab167453. Publication image and figure legend from Kumar, N., Theil, A. F., et al., 2022, Nat Commun, PubMed 35190564.

DDB2 binds sparse telomeric 8-oxoG independently of the DDB1-Cul4A-RBX1 E3 ligase.a Representative images showing recruitment of DDB2-mCherry to telomeric 8-oxoG in cells transfected with control, DDB1 or Cul4A siRNA. b Quantification of a. c, e DDB2-mCherry and GFP-DDB1 (c) or DDB2-mCherry and GFP-Cul4A (e) accumulation at 8-oxoG sites after dye (100 nM, 15 min) plus light (660 nm, 10 min) treatment. d, f Quantification of c and e respectively. g Western blot for DDB2 in U2OS-FAP-TRF1 cells treated with UVC, potassium bromate (KBrO3) or dye plus light at indicated doses. Independent experiments are represented by black circles. h Colocalization of DDB2-mCherry and GFP-Cul4A at damaged telomeres in U2OS-FAP-TRF1 cells transfected with control or OGG1 siRNA. i Quantification of h. Data (a-h) represents mean ± SEM from two independent experiments. 'n' represents the number of cells scored for each condition. One-way ANOVA (Sidak multiple comparison test) (b, i) was performed for statistical analysis : *p < < 0.05, **p < < 0.01, ***p < < 0.001, ns Not significant. Scale : 5 µm. Source data are provided as a Source Data file. (See also Supplementary Fig. 5).

• ICC/IF

CiteAb

Immunocytochemistry/ Immunofluorescence - Anti-mCherry antibody (AB167453)

mCherry immunocytochemistry-immunofluorescence using Anti-mCherry antibody ab167453. Publication image and figure legend from Lulla, V. & Firth, A. E., 2020, Nat Commun, PubMed 32792502.

Cellular localization, membrane topology, and multimerization of XP.a Huh7.5.1 cells were electroporated with pCAG-mCherry, pCAG-mCherry-XP, or pCAG-XP-mCherry. Representative confocal images of live cells stained for plasma membrane (WGA, green) and nuclei (Hoechst, blue); mCherry fluorescence is shown in red. HeLa cells were electroporated with pCAG-HA-XP, stained for plasma membrane (WGA, green), fixed, permeabilized and stained for XP with anti-HA antibody (red) and nuclei (Hoechst, blue). Images are averaged single plane scans. b HeLa cell lysates were fractionated and whole cell lysate (WCL), cytoplasmic (Cyto), membrane (Mem), and soluble nuclear (Nucl) fractions were analyzed by immunoblotting with antibodies to mCherry, tubulin, VDAC or Lamin A + C as indicated. See Supplementary Fig. 14 for complete images. c HeLa cells were electroporated with pCAG-XP-mCherry or pCAG-mCherry-XP. Cell surface mCherry on live cells was detected by incubation with anti-mCherry antibody, followed by staining with Alexa 488-labeled anti-rabbit IgG antibody and confocal microscopy. The images are averaged single plane scans. See Supplementary Fig. 15 for plasma membrane-permeabilized controls. A schematic representation of the observed membrane topologies is shown at right. d HeLa cells were electroporated with pCAG-XP-HA, fixed, permeabilized, and stained for XP (anti-HA, red), nuclei (Hoechst, blue), cis Golgi (anti-GM130, green), and trans Golgi (anti-TGN46, green). The images are averaged single plane scans. All scale bars are 10 µm (a,c,d). e Quantification of co-localization of XP-HA with TGN46 and GM130. The Pearson correlation coefficient was calculated for 12 images in each experiment. f Kyte-Doolittle hydropathy plots for HAstV XPs (see Supplementary Fig. 16 for individual plots). g C-terminal XP sequences for wt and 5L mutant HAstV1, and helical wheel representation of wt amino acids 92-109. h Huh7.5.1 cells were electroporated with wt or 5L mutant pCAG-Strep-XP or pCAG-HA-XP. At 16 hpe cells were lysed and subjected to immunoprecipitation using anti-HA magnetic beads. Presence of tagged proteins (5% of input and IP) was determined by western blotting using the indicated antibodies. See Supplementary Fig. 14 for complete images. Source data are provided as a Source Data file.

• IHC-Fr

CiteAb

Immunohistochemistry (Frozen sections) - Anti-mCherry antibody (AB167453)

mCherry immunohistochemistry (Frozen sections) using Anti-mCherry antibody ab167453.Publication image and figure legend from Stincic, T. L., Qiu, J., et al, 2021, Eneuro, PubMed 34281980.

Kiss1Cre-dependent ChR2-mCherry expressing neurons in the AVPV-PeN are also positive for kisspeptin. A, B1, C, Low-magnification, fluorescent images of Cre-driven ChR2-mCherry expression enhanced with ICC for mCherry. mCherry-labeled Kiss1 cells are distributed within the AVPV (A) and in a more narrow vertical band along the third ventricle (3V) from rostral to caudal PeN (B1, C). The section in B1 was also immunostained for kisspeptin (B2). D-G, Composite confocal images demonstrating that cells exhibiting Cre-driven mCherry expression are co-labeled by the Caraty kisspeptin antibody (green; arrows), but not all immunoreactive kisspeptin neurons also expressed ChR2-mCherry (arrow-head), likely because of incomplete coverage/infection by injected virus. AC, anterior commissure; AVPV, anteroventral periventricular nucleus; MnPO, median preoptic nucleus; MPO, medial preoptic nucleus; PeN, periventricular nucleus; OC, optic chiasm; 3V, third ventricle). Scale bars : 50 μm (A-C) and 10 μm (D-G).

• IHC-Fr

CiteAb

Immunohistochemistry (Frozen sections) - Anti-mCherry antibody (AB167453)

mCherry immunohistochemistry (frozen sections) using Anti-mCherry antibody ab167453. Publication image and figure legend from Stincic, T. L., Qiu, J., et al. 2021, Eneuro PubMed 34281980.

Interactions between GnRH and Kiss1 fibers in the ME. Confocal image montages of double label ICC that show Kiss1 fibers (ChR2-mCherry) originating from either the AVPV/PeN or ARH. GnRH fibers (green) enter the ARH laterally and along the 3V from the POA before entering the ME. A, Kiss1AVPV/Pen fibers are diffuse in the ARH and do not enter the ME. B, Expanded view of the dashed box from A. Few Kiss1AVPV/PeN fibers reach the ventral surface where GnRH fibers concentrate, which would suggest interactions are unlikely. C, Confocal image montage displays staining of Kiss1ARH cell bodies and projections in the cARH and ME. D, Single optical slice (1 μm thick) of the region outlined in C. GnRH fibers enter the ARH laterally (left) to briefly form a bundle (white arrow) that runs along the ventral surface before dispersing in the ME. D, Expanded view of the left white box in C. The occasional presence of yellow pixels and lack of black pixels between Kiss1ARH and GnRH fibers suggests the presence of interactions. E, F, Expanded view of the regions from the white boxes in D. As the GnRH fibers enter the ME the lack of black pixels between Kiss1ARH : : ChR2-mCherry and GnRH fibers and presence of yellow pixels suggest close contacts (white arrows) between fibers.

• IHC-Fr

CiteAb

Immunohistochemistry (Frozen sections) - Anti-mCherry antibody (AB167453)

mCherry immunohistochemistry (frozen sections) using Anti-mCherry antibody ab167453. Publication image and figure legend from Stincic, T. L., Qiu, J., et al., 2021, Eneuro PubMed 34281980.

Kiss1 fiber projections to AVP neurons in the SCN. A) Confocal image montage shows that Kiss1AVPV/PeN fibers (red) run along the 3V and surround the SCN. Double label ICC was used to visualize AVP neurons (green) which are primarily located in the shell of the SCN. a, Although practically no Kiss1AVPV/PeN fibers enter the SCN core, when viewing a single optical plane close contacts appear to be made with some of the most external AVP neurons (white arrows). B, Confocal image montage of the Kiss1ARH fibers in the rSCN. Few AVP cells bodies are present, but ascending projections can be seen running parallel to the 3V. b, Single optical slice centered on the region demarcated by the white box in B. A number of Kiss1ARH fibers pass through the SCN, potentially making close contact with AVP neurons or their fiber projections. However, the density of labeled AVP projections obscures somata and hinders analysis.

• IHC-Fr

CiteAb

Immunohistochemistry (Frozen sections) - Anti-mCherry antibody (AB167453)

mCherry immunohistochemistry (frozen sections) using Anti-mCherry antibody ab167453. Publication image and figure legend from Stincic, T. L., Qiu, J., et al., 2021, Eneuro, PubMed 34281980.

Interactions between GnRH and Kiss1 fibers in the ME. Confocal image montages of double label ICC that show Kiss1 fibers (ChR2-mCherry) originating from either the AVPV/PeN or ARH. GnRH fibers (green) enter the ARH laterally and along the 3V from the POA before entering the ME. A, Kiss1AVPV/Pen fibers are diffuse in the ARH and do not enter the ME. B, Expanded view of the dashed box from A. Few Kiss1AVPV/PeN fibers reach the ventral surface where GnRH fibers concentrate, which would suggest interactions are unlikely. C, Confocal image montage displays staining of Kiss1ARH cell bodies and projections in the cARH and ME. D, Single optical slice (1 μm thick) of the region outlined in C. GnRH fibers enter the ARH laterally (left) to briefly form a bundle (white arrow) that runs along the ventral surface before dispersing in the ME. D, Expanded view of the left white box in C. The occasional presence of yellow pixels and lack of black pixels between Kiss1ARH and GnRH fibers suggests the presence of interactions. E, F, Expanded view of the regions from the white boxes in D. As the GnRH fibers enter the ME the lack of black pixels between Kiss1ARH : : ChR2-mCherry and GnRH fibers and presence of yellow pixels suggest close contacts (white arrows) between fibers.

• WB

CiteAb

Western blot - Anti-mCherry antibody (AB167453)

mCherry western blotting using Anti-mCherry antibody ab167453. Publication image and figure legend from Sadhu, L., Tsopoulidis, N., et al., 2023, Elife, Pubmed 37162507.

Nuclear F-actin (NFA) formation induced by aphidicolin (APH) is not dependent on calcium signaling in CD4 T cells.(A) Dots represent the mean of each independent experiment, and the bar graph represents the mean of three experiments, showing % of cells forming NFA upon replication stress induction using increasing concentrations of APH. (B) Representative immunoblots show induction of phospho levels of DNA damage sensor CHK-1 in JNLA cells upon replication stress induction by APH (15 µm, APH) for 3 hr. Membranes were first probed with phospho-specific antibodies, followed by stripping and reprobing with antibodies against total protein and GAPDH. Immunoblots are representative of three independent experiments where the numbers indicated below the blots represent the mean ± SD intensity values of each condition compared to mCherry as control (set to 1). Intensity values from densitometric analysis for both phospho and total protein levels were normalized to GAPDH before further comparisons were done. (C) Single-cell tracking of 10 cells per condition (denoted by different colors for NTC, C5, or C5L KO) for the entire time frame of 5 hr post pretreatment with APH shows the APH-mediated NFA kinetics in KO and control JNLA cells. (D) shows relative comparison (fold change, FC) of cells forming either NFA or F-actin ring (AR), respectively, in control and KO JNLA cells upon two different modes of T cell activation, that is, activation with P/I or on anti-CD3/28-coated coverslips. Dots represent the mean of each independent experiment, and the bar graph represents the mean of three experiments with error bars calculated from mean ± SD of three independent experiments where at least 30 cells were analyzed per condition per experiment for NFA quantification and more than 100 cells per condition per experiment were analyzed for AR quantification. Statistical significance was calculated using one-way ANOVA (Kruskal-Wallis test) where ***p≤0.0002, ****p≤0.000021, and ns : not significant. (E, F) Scatter plots represent the number of filaments per cell (E) and the mean fluorescence intensity (MFI) per cell forming NFA (F) upon replication stress induction with APH comparing cells that express mCherry or nuclear-specific CAMBP4-mCherry in JNLA cells. 26 single cells were analyzed manually using Fiji from each of the experimental conditions. (G) Blocking of the calcium signaling pathway downstream of calmodulin using inhibitors STO609, KN93, KN62, and cyclosporin A (CsA), respectively, on JNLA cells, followed by induction of replication stress with APH does not impair the replication stress-mediated NFA burst observed. 30 cells/condition were analyzed in each experiment. Bar graph represents the mean from three independent experiments with each dot representing the mean from each independent experiment. Source data is avaialble at https : //doi.org/10.11588/data/YVYEO8.

false

• WB

CiteAb

Western blot - Anti-mCherry antibody (AB167453)

mCherry western blotting using Anti-mCherry antibody ab167453. Publication image and figure legend from Bodrug, T., Wilson-Kubalek, E. M., et al., 2020, Elife, PubMed 31958056.

Deletion of the kinesin-5 tail domain disrupts localization of the motor to the mitotic spindle in metaphase and anaphase.(A) Western blot for mCherry (mCh, green) and GAPDH (red) indicating the expression of FL-Eg5-mCherry (FL Eg5-mCh, green) and Eg5-δtail-mCherry (Eg5-δtail-mCh, green) in HeLa cells. Results are representative of three independent experiments. (B) Localization of mCh, FL-Eg5-mCh, and Eg5-δtail-mCh in HeLa cells arrested in metaphase via treatment with MG-132. FL-Eg5-mCh localized to spindle MTs. Tail deletion disrupted localization, and Eg5-δtail-mCh signal was distributed between spindle MTs and the cytoplasm. Scale bar 10 μm. Images are representative of three independent experiments. (C) Left panel, mCh fluorescence intensities of single cells used for quantification of localization to spindle MTs (n = 13-29 cells per transfection condition, three independent experiments). Right panel, the ratio of mCh fluorescence signal on the spindle to signal in the cytoplasm was significantly lower in fixed metaphase cells expressing Eg5-δtail-mCh compared to FL-Eg5-mCh, indicating reduced localization of Eg5-δtail-mCh to spindle MTs (n = 13-29 cells per transfection condition, three independent experiments, p values from ANOVA with Tukey's post hoc test). (D) Treatment of live HeLa cells expressing Eg5-mCh constructs and GFP-Tubulin with the Eg5 rigor inhibitor BRD-9876 resulted in a rapid (<1 min) increase in FL-Eg5-mCh signal on the spindle. Inhibitor treatment increased, but did not fully rescue, localization of Eg5-δtail-mCh to the spindle. Scale bar 10 μm. Images are representative of three independent experiments. (E) The ratio of mCh fluorescence signal on the spindle to signal in the cytoplasm rapidly increased after treatment with BRD-9876 in cells expressing FL-Eg5-mCh or Eg5-δtail-mCh. The spindle-to-cytoplasm intensity ratio of Eg5-δtail-mCh expressing cells never reached that of cells expressing FL-Eg5-mCh, indicating only partial rescue of motor localization with rigor inhibitor treatment. BRD-9876 treatment did not alter the ratio of mCh control cells (n = 7-13 cells per transfection condition, three independent experiments). (F) Deletion of the tail domain disrupted localization of Eg5 to the spindle in anaphase. Paired rows of images demonstrate the localization of FL-Eg5-mCh and Eg5-δtail-mCh as HeLa cells expressing GFP-tubulin transitioned from metaphase to anaphase. FL-Eg5-mCh signal was observed at the spindle throughout the metaphase to anaphase transition and the motor localized to the midzone after anaphase onset (see 4-6 min panels). Increased cytoplasmic and reduced spindle signal was observed in cells expressing Eg5-δtail-mCh throughout the metaphase to anaphase transition. Scale bar 10 μm. Images are representative of three independent experiments. (G) The ratio of mCh fluorescence signal on the spindle to signal in the cytoplasm was measured six minutes after anaphase onset. As in metaphase cells, localization of Eg5-δtail-mCh to the spindle was significantly reduced compared to FL-Eg5-mCh (n = 7-12 cells per transfection condition, three independent experiments, p values from ANOVA with Tukey's post hoc test). (H) The spindle-to-cytoplasm intensity ratio of cells expressing Eg5-δtail-mCh was lower than that of cells expressing FL-Eg5-mCh throughout the metaphase to anaphase transition, indicating a persistent localization defect caused by deletion of the tail domain (n = 7-12 cells per transfection condition, three independent experiments).

false

• WB

CiteAb

Western blot - Anti-mCherry antibody (AB167453)

mCherry western blotting using Anti-mCherry antibody ab167453. Publication image and figure legend from Jing, S., Gao, J., et al., 2024, Commun Biol, PubMed 38710927.

SUMOylation enhances Golgin45 protein stability by inhibiting TNKS1-dependent PARylation of Golgin45.a XAV939 treatment increases SUMO1-modified SUMOylation on Golgin45. HeLa cells were transiently transfected with mCherry-Golgin45 and SUMO1 or SUMO3 along with Ubc9 WT or DN, followed by either DMSO or XAV939 (a specific inhibitor of TNKS1/2). The cells were lysed and analyzed by western blotting using antibodies against the indicated proteins. b Relative levels of SUMO1 or SUMO3 conjugated mCherry-Golgin45 are presented as mean ± SD. Statistical analysis of relative SUMOylation was performed by one-way ANOVA with Tukey's multiple comparisons. n = 3 independent experiments. ns : not significant. ****p < < 0.0001. c, d Deletion of TNKS1 binding domain (δTBD) increases both SUMO1 and SUMO3 modified SUMOylation of Golgin45. After transfection with the indicate plasmids, HeLa cells were lysed and analyzed by western blotting using antibodies against the indicated proteins. e, f HeLa-Golgin45-KO cells stably expressing mCherry-Golgin45, mCherry-Golgin45-8KR mutant or mCherry-Golgin45-δTBD mutant were treated with cycloheximide (50 μg/ml) for the indicated times. Ectopically expressed WT and mutant mCherry-Golgin45 protein levels were analyzed by immunoblotting. Quantification of exogenous protein levels relative to GAPDH expression is shown in (f). Relative protein levels of mCherry-Golgin45 are presented as mean ± SD. Statistical analysis of relative protein level was performed by two-way ANOVA with Dunnett's post-hoc test for multiple comparisons. n = 3 independent experiments. ***p < < 0.001. g SUMOylation of mCherry-Golgin45 inhibits its PARylation. Hela cells transfected with indicate plasmids were lysed and immunoprecipitated using anti-RFP beads, and blotted with either anti-mCherry, anti-myc or anti-PAR. h Golgin45 PARylation is increased for Golgin45 8-KR mutant. HeLa cells expressing GST, GST-Golgin45 or GST-Golgin45-8KR were lysed and pulled down using GST beads, and analyzed by western blotting using anti-GST or anti-PAR antibodies. Representative blots are shown and experiments were repeated three times.

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