701064

antibody from Invitrogen Antibodies

Targeting: RPS6KB1 p70(S6K)-alpha, PS6K, S6K, S6K1, STK14A

Western blot (Supportive data in Antibodypedia)

Immunocytochemistry (Supportive data in Antibodypedia)

Flow cytometry (Supportive data in Antibodypedia)

Antibody Data

Product number

701064

Provider

Invitrogen Antibodies

Product name

Phospho-p70 S6 Kinase (Thr389) Recombinant Rabbit Monoclonal Antibody (B2H9L2)

Antibody type

Monoclonal

Antigen

Synthetic peptide

Description

Intact IgG appears on a non-reducing gel as ~150 kDa band and upon reduction generating a ~25 kDa light chain band and a ~50 kDa heavy chain.

Antibody clone number

B2H9L2

Concentration

0.5 mg/mL

Western blot

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Western blot analysis of p70S6K (pT389) was performed by loading 30 µg of untreated HeLa cells (lane 1) and HeLa cells treated with IGF-1 (150 ng/mL for 15 minutes) (lane 2) and U-87 MG lysates (lane 4) using Novex®NuPAGE®4-12% Bis-Tris gel (Product # NP0321BOX), XCell SureLock Electrophoresis System (Product # EI0002), and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Proteins were transferred to a PVDF membrane and blocked with 5% skim milk for 1 hour at room temperature. p70S6K (pT389) was detected at ~70 kDa using p70S6K (pT389) Recombinant Rabbit Monoclonal Antibody (Product # 701064) 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, 5). Detection was performed using an HRP-conjugated Goat anti-Rabbit secondary antibody (Product # G-21234) at a 1:5000 dilution and chemiluminescent detection was performed using Pierce™ ECL Western blotting Substrate (Product # 32106).

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Western blot analysis of Phospho-p70S6K Thr389 in whole cell extracts of serum-starved HeLa cells treated with IGF1 (150 ng/mL, 15 min) (lanes 1 and 2), and U87-MG cells (lanes 3 and 4) using a Phospho-p70S6K Thr389 recombinant rabbit monoclonal antibody (Product # 701064) at a dilution of 1 µg/mL. To confirm specificity, competition was performed by preincubation with the phosphopeptide to inhibit antibody binding (lanes 2 and 4). Samples were detected using chemiluminescence (ECL). Results show a band at ~70 kDa.

Immunocytochemistry

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunofluorescent analysis of Phospho-p70S6 Thr389 in serum-starved HeLa cells treated with insulin (100 ng/mL, 15 min) using a Phospho-p70S6 Thr389 recombinant rabbit monoclonal antibody (Product # 701064) followed by detection using an Alexa Fluor 488-conjugated goat anti-rabbit secondary antibody (green) (Image A). Nuclei were stained using DAPI (Image B) and actin stained with Alexa Fluor 594 phalloidin (red) (image C). Image D is a composite image showing cytoplasmic and nuclear localization of phosphorylated p70S6K and Image E is a composite image of cells showing competition with the phospho p70S6K (pT389) peptide.

Flow cytometry

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Flow cytometry analysis of p70S6K (pT389) was performed on U-87 MG cells. Cells were fixed with 70% ethanol for 10 minutes, permeabilized with 0. 25% Tritonª X-100 for 20 minutes, and blocked with 5% BSA for 1 hour at room temperature. Cells were labeled with ABfinityª p70S6K (pT389) recombinant rabbit monoclonal antibody (Product # 701064, red histogram) or with rabbit isotype control (pink histogram) at a dilution of 1:250 in 2.5% BSA. After incubation at room temperature for 3 hours, the cells were labeled with Alexa Fluor¨ 488 goat anti-Rabbit Secondary antibody (Product # A11008) at a dilution of 1:400 for 30 minutes at room temperature. The representative 10,000 cells were acquired and analyzed for each sample using an Attune¨ Acoustic Focusing Cytometer. The purple histogram represents unstained control cells and the green histogram represents no-primary-antibody control.