Anti rab11fip2

The cells were fixed with 4% paraformaldehyde for 30 min and then treated with 0.5% Triton X-100 for 10 min, followed by 30 min of blocking with 1% BSA at room temperature. The primary antibodies anti-RAB11FIP2 (Abcam, UK, #ab180504), anti-AKT (Proteintech, USA, #10176–2-AP), and anti-p-AKT (Ser473) (Proteintech, USA, #664441-Ig) were added, and the wells were incubated overnight at 4°C. The next steps were the same as described previously (20 (link)).

Proteins were extracted with RIPA lysis buffer (KeyGen Biotech, Nanjing, China) and quantified by using a bicinchoninic acid protein quantification kit (KeyGen Biotech, Nanjing, China). Proteins were separated by SDS-PAGE and then transferred onto PVDF membranes (Millipore, Bedford, USA), followed by blocking with PBST solution containing 5% skim milk or 5% BSA for 1 h. Subsequently, the primary antibody was added, and the membranes were incubated at 4°C overnight, followed by a 1-h incubation with the corresponding secondary antibody at room temperature. Finally, Pierce ECL Western Blotting Substrate (FDbio Science, China) was used for the chemiluminescence assay. The following primary antibodies were used: anti-RAB11FIP2 (Abcam, UK, #ab180504), anti-AKT (Proteintech, USA, #10176–2-AP), anti-p-AKT (Ser473) (Proteintech, USA, #664441-Ig), anti-PI3K (Abcam, UK, #ab140307), anti-p-PI3K (Tyr607) (Abcam, UK, #ab182651) and anti-GAPDH (Proteintech, USA, #10494–1-AP). PI3K inhibitor LY294002 (Beyotime, Shanghai, China, #S1737) was used at a concentration (50 umol/L).