Human/Mouse Phospho-PRAS40 (T246) Antibody

Detection of Human and Mouse Phospho-PRAS40 (T246) by Western Blot. Western blot shows lysates of MCF-7 human breast cancer cell line and NIH-3T3 mouse embryonic fibroblast cell line untreated (-) or treated (+) with 100 ng/mL Recombinant Human IGF-I (Catalog # 291-G1) for 20 minutes and 10 ng/mL Recombinant Human PDGF-BB (Catalog # 220-BB) for 5 minutes. PVDF membrane was probed with 0.5 µg/mL of Mouse Anti-Human Phospho-PRAS40 (T246) Monoclonal Antibody (Catalog # MAB6890) followed by HRP-conjugated Anti-Mouse IgG Secondary Antibody (Catalog # HAF018). A specific band was detected for Phospho-PRAS40 (T246) at approximately 40 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.

Phospho-PRAS40 (T246) in MCF‑7 Human Cell Line. PRAS40 phosphorylated at T246 was detected in immersion fixed MCF-7 human breast cancer cell line using Mouse Anti-Human/Mouse Phospho-PRAS40 (T246) Monoclonal Antibody (Catalog # MAB6890) at 25 µg/mL for 3 hours at room temperature. Cells were stained using the NorthernLights™ 557-conjugated Anti-Mouse IgG Secondary Antibody (red; Catalog # NL007) and counterstained with DAPI (blue). Specific staining was localized to cytoplasm. View our protocol for Fluorescent ICC Staining of Cells on Coverslips.

Detection of Human PRAS40 by Western Blot PIK3R2 depletion, but not PI3K inhibitors, induces stable PI3K pathway inhibitionA. Tumor xenografts were obtained using H226, CaLu-1 and H520 lung SQCC cells expressing inducible PIK3R1 or PIK3R2 shRNA. When tumors reached ~50 mm3, mice were treated with doxycycline in drinking water (2 mg/ml); they were sacrificed when tumors began to diminish. Normalized tumor extracts were examined by WB with indicated antibodies. B. H226, CaLu-1 and H520 cells were cultured in exponential growth were incubated with: vehicle (DMSO, 1:103 V:V), Ly294002 (5 μM), PIK75 (200 nM), TGX221 (30 μM), or PIK75 (200 nM) plus TGX221 (30 μM) for the last 1 h of culture or during 48 h; extracts were tested in WB. Mr indicates relative mobility. In both A and B, the pAkt or pPRAS40 signal was measured and normalized to the loading control (Akt or tubulin, respectively), and compared to that in controls (DMSO, considered 100%). Graphs show percentage of signal compared to maximal as mean ± SD at 48h of treatment. Arrows on the right side of bars show the percentage of signal compared to maximal detected after 1h of treatment.* P <0.05; **P <0.01; unpaired Student's t test with Welch correction. Image collected and cropped by CiteAb from the following publication (https://www.oncotarget.com/lookup/doi/10.18632/oncotarget.13195), licensed under a CC-BY license. Not internally tested by R&D Systems.