Pi3k p85 antibody

Recombinant Human CXCL12 and anti-human CXCL12 antibody were purchased by R&D system Inc. (Minneapolis, MN, USA). LY294002 (PI3K inhibitor) was ordered from Cell Signaling Technology (Beverly, MA, USA). The monoclonal antibodies (mAbs) included PTEN antibody, phospho-PTEN (ser380) antibody, Akt antibody, phospho-Akt (ser473), PI3K p85 antibody, phospho-PI3K p85 (Tyr 458) /p55 (Tyr199) antibody were provided by Cell Signaling Technology.

After washing with cold PBS, cells were homogenized in RIPA lysis buffer (Beyotime, Jiangsu, China). Total crude protein (60 mg per well) was separated on 10% SDS‐PAGE gels. Then, it was transferred to PVDF membranes (Millipore, MA, USA). After washing for 10 minutes with TBST (0.1% Tween‐20, TBS) three times, the membranes were placed into blocking buffer (TBST with 5% non‐fat milk) for 1 hour at room temperature. And then, the membranes were incubated with primary antibodies against HO‐1 (0.5 μg/mL, 3391‐100; BioVision, San Francisco Bay Area, California, USA), PI3K p85 antibody (1:1000, #4292; Cell Signaling Technology, Beverly, Mass, USA), Akt (1:1000, #9272; Cell Signaling Technology), p‐Akt (1:1000, #9271; Cell Signaling Technology), LC3 (0.5 μg/mL, ab48394; Abcam, London, UK), Beclin‐1 (0.5 μg/mL, ab62557; Abcam) and β‐actin (1:2000, ab8227; Abcam) overnight at 4°C. The next day, after washing for 15 minutes with TBST (0.1% Tween‐20, TBS) four times, membranes were incubated with goat anti‐rabbit IgG (1:5000, ab6721; Abcam) for 1 hour at 37°C. Finally, it was visualized by ECL‐PLUS reagents (Beyotime). β‐actin was used for normalization of protein expression.