2.7. Protein Isolation and Western Blot Analysis

Total protein was recovered from pig backfat and 3T3-L1 cells using an ice-cold lysis buffer containing PMSF (phenylmethylsulfonyl fluoride). The samples were centrifuged at 12,000× g for 30 min at 4 °C. The concentration of total protein was determined using a Braford protein assay kit (Tiangen- PA102). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to separate 30 µg of total protein and transfer it to polyvinyl difluoride (PVDF) membranes (Millipore, Boston, MA, USA). Anti-PIK3R1 (ab86714, Abcam, Cambridge, United Kingdom) and anti-tubulin (ab15246, Abcam) primary antibodies were used to probe the membranes, followed by an appropriate secondary antibody (anti-rabbit-HRP (ab97051, Abcam) or anti-mouse-HRP (ab205719, Abcam)). Before usage, the primary antibodies against PIK3R1 and tubulin were diluted to 1:3000 and 1:5000, respectively. Secondary antibodies were diluted to a concentration of 1:5000.