Anti-p70 S6 Kinase (phospho T421 + S424) antibody [RP23040018]

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-p70 S6 Kinase (phospho T421 + S424) antibody [RP23040018] (AB308099)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0. 25% Triton X-100 permeabilized 70% confluent log phase U-2 OS cells labeling p70 S6 Kinase (phospho T421 + S424) with ab308099 at 1/200 dilution, followed by Alexa Fluor® 488 Goat anti-Rabbit IgG secondary antibody at 1/400 dilution (Panel a : green). Nuclei (Panel b : blue) were stained with DAPI. F-actin (Panel c : red) was stained with Alexa Fluor® 594 phalloidin. Panel d is a merged image showing cytoplasmic localization respectively. Panel e shows competition with the p70 S6 Kinase (phospho T421 + S424) peptide. The images were captured at 20X magnification.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-p70 S6 Kinase (phospho T421 + S424) antibody [RP23040018] (AB308099)

Immunofluorescent analysis of 4% paraformaldehyde-fixed serum-starved HeLa cells treated with insulin (100 ng/mL, 15 min) labeling p70 S6 Kinase (phospho T421 + S424) with ab308099, followed by Alexa Fluor® 488 Goat anti-Rabbit IgG secondary antibody (green). Imaging shows cytoplasmic and nuclear localization of p70 S6 Kinase (phospho T421 + S424) nuclei (blue) were stained with DAPI. F-actin (red) was stained with Alexa Fluor® 594 phalloidin. Images A and B are a composite images. Image C shows results of a competition assay using the p70 S6 Kinase (phospho T421 + S424) peptide.

• WB

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Western blot - Anti-p70 S6 Kinase (phospho T421 + S424) antibody [RP23040018] (AB308099)

Western blot analysis of p70 S6 Kinase (phospho T421 + S424) in whole cell extracts of serum-starved HeLa cells treated with IGF1 (150 ng/mL, 15 min) using ab308099 at a dilution of 1 µg/mL. To confirm specificity, competition was performed by preincubation with the phosphopeptide to inhibit antibody binding (lane 2). Results show a band at ~70 kDa.

All lanes:

Western blot - Anti-p70 S6 Kinase (phospho T421 + S424) antibody [RP23040018] (ab308099) at 1 µg/mL

Lane 1:

serum-starved HeLa cells treated with IGF1

Lane 2:

Preincubation of HeLa cell lysate with phosphopeptide and IGF-1

Predicted band size: 59 kDa

Observed band size: 70 kDa

false

• WB

Supplier Data

Western blot - Anti-p70 S6 Kinase (phospho T421 + S424) antibody [RP23040018] (AB308099)

Western blot analysis of p70 S6 Kinase (phospho T421 + S424) was performed by loading 20 µg of untreated HeLa (lane 1) and HeLa treated with IGF1 (150 ng/mL for 15 minutes) cell lysates (lane 2) using a 4-12% Bis-Tris gel electrophoresis system, and pre-stained protein standard. Proteins were transferred to a nitrocellulose membrane and blocked with 5% skim milk for 1 hour at room temperature. p70 S6 Kinase (phospho T421 + S424) was detected at ~70 kDa using ab308099 at a 1 : 1000 dilution in 2.5% skim milk at 4°C overnight on a rocking platform. To confirm specificity, competition was performed with the phosphopeptide (10 µg/mL) (lane 3). Detection was performed using an HRP-conjugated Goat anti-Rabbit secondary antibody at a 1 : 5000 dilution and chemiluminescent detection was performed (ECL)

All lanes:

Western blot - Anti-p70 S6 Kinase (phospho T421 + S424) antibody [RP23040018] (ab308099) at 1/1000 dilution

Lane 1:

Untreated HeLa cell lysate at 20 µg

Lane 2:

HeLa cell lysate treated with IGF1 at 20 µg

Lane 3:

Hela cell lysate treated with IGF-1 and phosphopeptide at 10 µg/mL

Secondary

All lanes:

HRP-conjugated Goat anti-Rabbit secondary antibody at 1/5000 dilution

Predicted band size: 59 kDa

Observed band size: 70 kDa

true