Anti-IKB alpha (phospho S32) antibody [EPR3148]

• IP

Unknown

Immunoprecipitation - Anti-IKB alpha (phospho S32) antibody [EPR3148] (AB92700)

Purified ab92700 at 1 : 20 dilution (1μg) immunoprecipitating IKB alpha in HeLa treated with 20ng/mL TNF-alpha for 60 minutes whole cell lysate.

Lane 1 (input) : HeLa (Human cervix adenocarcinoma epithelial cell) treated with 20ng/mL TNF-alpha for 60 minutes whole cell lysate 10μg.
Lane 2 (+) : ab92700 + HeLa treated with 20ng/mL TNF-alpha for 60 minutes whole cell lysate.
Lane 3 (-) : Rabbit monoclonal IgG (ab172730) instead of ab92700 in HeLa treated with 20ng/mL TNF-alpha for 60 minutes whole cell lysate.

Blocking Buffer and concentration : 5% NFDM/TBST.
Diluting buffer and concentration : 5% NFDM/TBST.
Observed band size : 36 kDa

All lanes:

Immunoprecipitation - Anti-IKB alpha (phospho S32) antibody [EPR3148] (ab92700)

Predicted band size: 35 kDa

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• WB

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Western blot - Anti-IKB alpha (phospho S32) antibody [EPR3148] (AB92700)

Blocking/Diluting buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-IKB alpha (phospho S32) antibody [EPR3148] (ab92700) at 1/1000 dilution

Lane 1:

MCF7 (Human breast adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

MCF7 (Human breast adenocarcinoma epithelial cell) treated with 20 ng/mL TNF-alpha for 8 hours whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 35 kDa

Observed band size: 36 kDa

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• WB

Lab

Western blot - Anti-IKB alpha (phospho S32) antibody [EPR3148] (AB92700)

Blocking/Diluting buffer and concentration 5% NFDM/TBST

All lanes:

Western blot - Anti-IKB alpha (phospho S32) antibody [EPR3148] (ab92700) at 1/500 dilution

Lane 1:

HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysates at 15 µg

Lane 2:

HeLa (Human cervix adenocarcinoma epithelial cell) treated with TNF-α at 20 ng/mL for 5 minutes. Whole cell lysates at 15 µg

Lane 3:

HeLa treated with TNF-α at 20 ng/mL for 5 minutes. Whole cell lysates. Then the membrane was incubated with phosphatase. at 15 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 35 kDa

Observed band size: 36 kDa

false

Exposure time: 3min

• WB

Unknown

Western blot - Anti-IKB alpha (phospho S32) antibody [EPR3148] (AB92700)

Blocking/Diluting buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-IKB alpha (phospho S32) antibody [EPR3148] (ab92700) at 1/1000 dilution

Lane 1:

HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate at 15 µg

Lane 2:

HeLa (Human cervix adenocarcinoma epithelial cell) treated with 20 ng/mL TNF-alpha for 5 minutes whole cell lysate at 15 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 35 kDa

Observed band size: 36 kDa

false

• WB

Supplier Data

Western blot - Anti-IKB alpha (phospho S32) antibody [EPR3148] (AB92700)

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

All lanes:

Western blot - Anti-IKB alpha (phospho S32) antibody [EPR3148] (ab92700) at 1/1000 dilution

Lane 1:

Untreated Raw264.7 (Mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysate at 20 µg

Lane 2:

Raw264.7 (Mouse Abelson murine leukemia virus-induced tumor macrophage) treated with 50 ng/mL TNF-a and 300 ng/ml BFA for 24 hours whole cell lysate at 20 µg

Lane 3:

Raw264.7 (Mouse Abelson murine leukemia virus-induced tumor macrophage) treated with 50 ng/mL TNF-a and 300 ng/ml BFA for 24 hours whole cell lysate.Then the membrane was incubated with alkaline phosphatase at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 35 kDa

Observed band size: 36 kDa

false

Exposure time: 180s

• WB

Supplier Data

Western blot - Anti-IKB alpha (phospho S32) antibody [EPR3148] (AB92700)

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

All lanes:

Western blot - Anti-IKB alpha (phospho S32) antibody [EPR3148] (ab92700) at 1/1000 dilution

Lane 1:

Untreated C6 (Rat glial tumor glial cell) whole cell lysate at 20 µg

Lane 2:

C6 (Rat glial tumor glial cell) treated with 100 ng/mL Calyculin A for 30 minutes whole cell lysate at 20 µg

Lane 3:

C6 (Rat glial tumor glial cell) treated with 100 ng/mL Calyculin A for 30 minutes whole cell lysate. Then the membrane was incubated with alkaline phosphatase at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 35 kDa

Observed band size: 36 kDa

false

Exposure time: 20s

• Dot

Unknown

Dot Blot - Anti-IKB alpha (phospho S32) antibody [EPR3148] (AB92700)

Dot blot analysis of IKB alpha (phospho S32) phospho peptide (Lane 1) and IKB alpha non-phospho peptide (Lane 2) labeling IKB alpha (phospho S32) with unpurified ab92700 at a dilution of 1/1000. ab97051 (Peroxidase conjugated goat anti-rabbit IgG) (H+L) at 1/10000 was used as the secondary antibody.

Blocking and diluting buffer : 5% NFDM/TBST.

Exposure time : 3 minutes.

• WB

CiteAb

Western blot - Anti-IKB alpha (phospho S32) antibody [EPR3148] (AB92700)

Western Blotting using Anti-IKB alpha (phospho S32) antibody [EPR3148], ab92700. Publication image from Omer, A. et al., 2020, Nat Commun, 33020468. Legend direct from paper.

The G3BP1 inhibitor, EGCG, recapitulates SASP inhibition associated with G3BP1 loss.a (left) Cell lysates from proliferative and 8-day post-ionizing radiation (+IR) WI-38 cells treated or not with EGCG (40 µM) were subjected to western blot analysis against indicated proteins. (right) Quantifications represent a mean of relative protein levels from three independent experiments ± s.e.m (two-way ANOVA, Fisher’s LSD). b RNA was extracted from cells during proliferative stage (PRO) and 8-day post-ionizing radiation (+IR) from WI-38 cells treated with or without (NT) increasing concentrations of EGCG (10, 20, and 40 µM) and assayed by RT-qPCR using primers against the indicated mRNAs. The data are relative to PRO cells and are a mean of three independent experiments ± s.e.m (one-way ANOVA, Fisher’s LSD). c Conditioned media from proliferative (PRO) cells and 8-day post-ionizing radiation (SEN) WI-38 cells treated with or without 40 µM EGCG. The heatmap indicates the fold change in comparison to the control PRO, SEN, and SEN treated with 40 µM EGCG. Relative expression levels per replicate and average fold change differences are shown as indicated in heatmap. The data are representative of three independent experiments. d RNA was extracted from cells treated with siRNA targeting G3BP1 (siG3BP1) or scrambled control (siCTL) during proliferative stage (PRO) and 8-day post-ionizing radiation (+IR) from WI-38 cells in the presence or absence (NT) of EGCG (40µM). RT-qPCR was then performed using primers against indicated mRNAs. The data are a mean of three independent experiments ± s.e.m (two-way ANOVA, Fisher’s LSD, exact <0.001 p-values from left to right : (top) 0.0001, 0.0001, 0.0001, 0.0011, (bottom) 0.0001, 0.0001, 0.0001, 0.0004, 0.0001, 0.0001). Source Data for panels (a), (b), and (d) are provided in the Source Data File. Raw data for (c) is provided in Supplementary Data 3.

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