Anti pi3k p85

Western blot (WB) was performed as reported previously 43 (link). The primary antibodies included anti-CD44, anti-CD133, anti-NANOG, anti-OCT4, anti-SOX2, anti-BMI1, anti-MDK, anti-PI3K-p85, anti-p-PI3K-p85, anti-AKT, anti-p-AKT and anti-ACTIN (diluted 1:1000, Cell Signaling Technology, USA). ACTIN was used as an internal control.

3T3-L1 adipocytes were seeded onto Matrigel-coated eight-well microslides (Ibidi). Forty-eight hours later, cells were serum-starved for 2 hr and then exposed to DMSO, 10 μM MK2206, or 10 μM GDC0068 for 5 min, followed by 1 nM insulin for 10 min. The coverslips were then briefly immersed in ice-cold PBS and fixed with 4% paraformaldehyde in PBS at room temperature for 15 min. Cells were then washed twice with room temperature PBS and quenched with 200 mM glycine for 10 min. Cells were then blocked and permeabilised with 2% BSA/0.1% saponin in PBS for 30 min. Cells were incubated with the anti-PI3K p85 (Cell Signaling Technology CST4257S) primary antibody (1:100 in 2% BSA/0.1% saponin in PBS) for 1 hr at room temperature. Cells were then washed with 2% BSA/0.1% saponin in PBS three times, and then incubated with anti-rabbit-IgG conjugated to Alexa Fluor 555 (1:200 in 2% BSA/0.1% saponin in PBS) at room temperature for 1 hr in the dark. Cells were then washed five more with PBS and then stored and imaged in 5% glycerol/2.5% 1,4-diazabicyclo[2.2.2]octane in PBS. Images were acquired using the Nikon Ti-LAPP H-TIRF module angled to image ∼90 nm into cells. To quantify relative changes in PM PI3K p85, for each cell we measured the median pixel intensity (corrected for background) using Fiji (Schindelin et al., 2012 (link)).

Total cell or tissue proteins were extracted using RIPA lysate (Beyotime, Shanghai, China). Proteins were separated using 10% SDS‐PAGE and transferred onto PVDF membranes (Millipore, Massachusetts, USA). After being blocked at room temperature for 2 h, the membranes were incubated with primary anti-TMEM119 (1:1000; Proteintech, Chicago, USA), anti-PDGFRB (1:1000; Proteintech, Chicago, USA), anti-PI3K p85 (1:1000; Cell Signaling Technology, Danvers, USA), anti-p-PI3K p85 (1:1000; Cell Signaling Technology, Danvers, USA), anti-AKT (1:1000; Cell Signaling Technology, Danvers, USA), anti-p-AKT (1:1000; Cell Signaling Technology, Danvers, USA), anti-mTOR (1:1000; Cell Signaling Technology, Danvers, USA), anti-p-mTOR (Ser2448) (1:1000; Cell Signaling Technology, Danvers, USA) or anti-GAPDH (1:3000; Proteintech, Chicago, USA) antibodies. The membranes were then washed and incubated with secondary antibodies. Protein bands were detected with enhanced chemiluminescence (Thermo Scientific, Carlsbad, USA).