Anti phospho pi3k tyr458

Cells and tissues were first harvested and processed to obtain total protein, which was quantified using the BCA Protein Assay Kit (Thermo Fisher Scientific, MA). Small quantities of proteins were then separated by migration through the sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) before being transferred onto 0.45-mm polyvinylidene difluoride (PVDF) membranes. The membranes were then blocked to prevent nonspecific binding and maintained in primary antibody solutions overnight (4°C). Incubation with secondary antibodies was performed at a temperature of 25°C and followed by washing steps (three washings). Detection was done with ECL TM Western Blotting Detection reagents, and the blots were detected using an enhanced chemiluminescence detection system (Bio-Rad). Images were captured and analyzed using Image J. The following primary antibodies were used in this study: anti-AKT (1:1000, 9272, Cell Signaling, MA), anti-phospho-AKT (Ser473) (1:1000, 9271, Cell Signaling, MA), anti-PI3K (1:1000, 4255, Cell Signaling, MA), anti-phospho-PI3K (Tyr458) (1:1000, 4228; Cell Signaling, MA), anti-ERK (1:1000, 9102; Cell Signaling, MA), anti-phospho-ERK (Thr202/Tyr204) (1:1000, 9101, Cell Signaling, MA), anti-GSTO1 (1:1000, ab266277, Abcam, MA), anti-GSTM1 (1:1000, ab178684, Abcam, MA), anti-TTR (1:1000, 11891-1-AP, proteintech,MA), and anti-β-actin (1:1000, ab8226, Abcam, UK).