Anti-NFkB p105 / p50 antibody [E381]

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NFkB p105 / p50 antibody [E381] (AB32360)

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.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NFkB p105 / p50 antibody [E381] (AB32360)

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.

• WB

Lab

Western blot - Anti-NFkB p105 / p50 antibody [E381] (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 HeLa 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] (ab32360) 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

Unknown

Western blot - Anti-NFkB p105 / p50 antibody [E381] (AB32360)

Blocking and dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-NFkB p105 / p50 antibody [E381] (ab32360) at 1/50000 dilution

All lanes:

MCF-7 cell lysate at 10 µg

Secondary

All lanes:

Peroxidase conjugated goat anti-rabbit IgG (H+L) at 1/1000 dilution

Predicted band size: 105 kDa

Observed band size: 105 kDa,50 kDa

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• WB

Unknown

Western blot - Anti-NFkB p105 / p50 antibody [E381] (AB32360)

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.

All lanes:

Western blot - Anti-NFkB p105 / p50 antibody [E381] (ab32360)

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

Unknown

Western blot - Anti-NFkB p105 / p50 antibody [E381] (AB32360)

Blocking and dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-NFkB p105 / p50 antibody [E381] (ab32360) at 1/10000 dilution

All lanes:

HeLa cell lysate at 20 µg

Secondary

All lanes:

Peroxidase conjugated goat anti-rabbit IgG (H+L) at 1/1000 dilution

Predicted band size: 105 kDa

Observed band size: 105 kDa,50 kDa

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• WB

Unknown

Western blot - Anti-NFkB p105 / p50 antibody [E381] (AB32360)

Blocking and dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-NFkB p105 / p50 antibody [E381] (ab32360) at 1/10000 dilution

Lane 1:

PC-12 cell lysate at 20 µg

Lane 2:

NIH/3T3 cell lysate at 20 µg

Secondary

All lanes:

Peroxidase conjugated goat anti-rabbit IgG (H+L) at 1/1000 dilution

Predicted band size: 105 kDa

Observed band size: 105 kDa,50 kDa

false

• WB

Unknown

Western blot - Anti-NFkB p105 / p50 antibody [E381] (AB32360)

All lanes:

Western blot - Anti-NFkB p105 / p50 antibody [E381] (ab32360) at 1/5000 dilution

All lanes:

HeLa cell lysate

Predicted band size: 105 kDa

Observed band size: 105 kDa,50 kDa

false

• WB

AbReview64956****

Western blot - Anti-NFkB p105 / p50 antibody [E381] (AB32360)

All lanes:

Western blot - Anti-NFkB p105 / p50 antibody [E381] (ab32360) at 1/1000 dilution

All lanes:

mouse neural stem cell lysate at 40 µg

Secondary

All lanes:

Alexa Fluor 790 Goat fluorescent secondary at 1/10000 dilution

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Exposure time: 1hr

This image is courtesy of an anonymous Abreview

• WB

CiteAb

Western blot - Anti-NFkB p105 / p50 antibody [E381] (AB32360)

Western Blotting using Anti-NFkB p105 / p50 antibody [E381], ab32360. Publication image from Galle, P. R. et al., 2017, Nat Commun, 28452361. Legend direct from paper.

Bcl-3 prevents p50 binding to the Foxp3 and CTLA-4 promoter.(a) Western blot analysis of Bcl-3 and the NF-κB family members p50, p52 and p65 in nuclear versus whole extract of Bcl-3TOE and littermate controls using Tregs either unstimulated (left) or restimulated for 2 h with PMA/Ionomycin (right). One representative blot from two independent experiments is shown. (b) Immunoprecipitation with Bcl-3 antibodies using protein extracts from in vitro differentiated Tregs, restimulated with PMA/Ionomycin for 4 h and followed by immunoblot analysis with antibodies against Bcl-3 or p50. One representative blot from two independent experiments is shown. (c) Pull-down experiments with DNA probes from promoters of IL2Ra, Foxp3 CN2 (from top to bottowm) with protein extracts from Tregs to which Bcl-3 or empty vector overexpressing lysates were added. One out of two experiments is demonstrated. (d) CHiP assay using p50 and isotope control antibodies antibodies were performed in in vitro differentiated Tregs from Bcl3TOE mice and littermate controls. Primer were designed as indicated for two sites within the CTLA-4 and Foxp3 Promoter region. One of two representative biological examples is shown.

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• WB

CiteAb

Western blot - Anti-NFkB p105 / p50 antibody [E381] (AB32360)

Western Blotting using Anti-NFkB p105 / p50 antibody [E381], ab32360. Publication image from Galle, P. R. et al., 2017, Nat Commun, 28452361. Legend direct from paper.

Bcl-3 inhibits Foxp3 promoter activity via direct binding to p50.(a) Reporter assays with constructs containing the endogenous Foxp3 promoter, using in vitro differentiated Tregs restimulated with PMA/Ionomycin from Bcl-3TOE mice. The Luciferase reads from the pGL4 plasmid that contained the Foxp3 promoter or empty were normalized to a Renilla transfection control plasmid. One representative out of two independent experiments with four parallel transfections is shown. Mean±s.e.m. ***P<0.001 using unpaired Student's t-test. (b) Immunoprecipitations using nuclear extracts from HEK293T cells transfected with Bcl-3 wt and Bcl-3 mutant co-transfected with p50 were performed, followed by immune blot analysis using Bcl-3 and p50 antibodies. (c) Reporter assays for Foxp3 gene activity as described in a with in vitro expanded T cells transfected with Bcl-3 wt and Bcl-3-p50 mutant constructs. Normalization was performed as in a. One representative out of two independent experiments with four parallel transfections is shown. Mean±s.e.m. **P<0.01 and ***P<0.001 using unpaired Student's t-test; s>3.

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