Anti-NFkB p105 / p50 antibody [E381] - BSA and Azide free

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NFkB p105 / p50 antibody [E381] - BSA and Azide free (AB220803)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human bladder carcinoma tissue labelling NFkB p105 / p50 with purified ab32360 at 1/250. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32360).

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NFkB p105 / p50 antibody [E381] - BSA and Azide free (AB220803)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human prostate carcinoma tissue labelling NFkB p105 / p50 with unpurified ab32360 at a dilution of 1/250.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32360).

• WB

Lab

Western blot - Anti-NFkB p105 / p50 antibody [E381] - BSA and Azide free (AB220803)

Lanes 1 - 4 : Merged signal (red and green). Green - ab32360 observed at 120, 50 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab32360 was shown to specifically react with NFkB p105 / p50 when NFkB p105 / p50 knockout samples were used. Wild-type and NFkB p105 / p50 knockout samples were subjected to SDS-PAGE. ab32360 and ab8245 (loading control to GAPDH) were diluted at 1/1000 and 1/10 000 respectively and and incubated overnight at 4°C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32360).

All lanes:

Western blot - Anti-NFkB p105 / p50 antibody [E381] - BSA and Azide free (ab220803)

Lane 1:

Wild-type HAP1 cell lysate at 20 µg

Lane 2:

NFkB p105 / p50 knockout HAP1 cell lysate at 20 µg

Lane 3:

HeLa cell lysate at 20 µg

Lane 4:

Jurkat cell lysate at 20 µg

Predicted band size: 105 kDa

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• WB

Lab

Western blot - Anti-NFkB p105 / p50 antibody [E381] - BSA and Azide free (AB220803)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32360).

Lanes 1- 2 : Merged signal (red and green). Green - ab32360 observed at 105 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

ab32360 was shown to react with NFkB p105 / p50 in wild-type HeLa cells in western blot. The band observed in CRISPR/Cas9 edited cell line ab264823 (CRISPR/Cas9 edited cell lysate ab257003) lane below 105kDa may represent truncated forms and cleaved fragments. This has not been investigated further. Wild-type HeLa and NFKB1 CRISPR/Cas9 edited HeLa 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. ab32360 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^®800CW) preadsorbed (ab216773) and 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-NFkB p105 / p50 antibody [E381] (<a href='/en-us/products/primary-antibodies/nfkb-p105-p50-antibody-e381-ab32360'>ab32360</a>) at 1/1000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

NFKB1 CRISPR/Cas9 edited HeLa cell lysate at 20 µg

Predicted band size: 105 kDa

Observed band size: 105 kDa

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• WB

CiteAb

Western blot - Anti-NFkB p105 / p50 antibody [E381] - BSA and Azide free (AB220803)

NFkB p105 / p50 western blot using anti-NFkB p105 / p50 antibody [E381] - BSA and Azide free ab220803. Publication image and figure legend from Xiao, X., Shi, X., et al., 2015, Nat Commun, PubMed 26365427.

ab220803 was used in this publication in western blot. This may not be the same as the application(s) guaranteed by Abcam. For a full list of applications guaranteed by Abcam for ab220803 please see the product overview.

Role of p50 in recruitment of histone deacetylases to the Foxp3 locus to inhibit Foxp3 expression.(a) Schematic diagram depicting Foxp3 locus structure and consensus κB binding sites (boxed) in the Foxp3 promoter and CNS1–2 regions. (b) ChIP analysis of p50 enrichment at the Foxp3 promoter and CNS1–2 sites in naive CD4+ cells left untreated (Unstim) or activated under iTreg-polarizing conditions in the presence of DTA-1. Data represent mean values±s.d. (n=4). (c) Co-immunoprecipitation analysis of p50 in naive CD4+ T cells activated as in (b). Anti-p50 or control IgG immunoprecipitates (IP) were subjected to immunoblot analysis (IB) using anti-HDAC1 and anti-Sirt1 antibodies. Data are representative of three independent experiments. (d) ChIP analysis of HDAC1 binding to the Foxp3 promoter and CNS1–2 sites in naive CD4+ cells stimulated under iTreg-polarizing conditions in the presence of DTA-1 or IgG. Data represent mean values±s.d. (n=3). (e,f) Flow cytometry analysis of Foxp3 expression in naive CD4+ T cells activated as in (d) for 3 days in the presence of HDAC1 inhibitor CI-994 (CI, 1 μM) or Sirt1 inhibitor EX-527 ( EX, 0.5 μM). Numbers in the quadrants indicate the percentage of Foxp3+ cells (e). (f) Plots shows mean±s.d. of Foxp3+ T cells from 3 experiments with duplicate culture. P values were determined by Student's t-test (*p<0.05).

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