Molecular imager chemidoc xsr gel imaging system

Referring to the previous description (32 (link)), protein extraction and immunoblotting were performed. The antibodies directed against the following proteins, including PI3K, p-PI3K, AKT, p-AKT, and β-actin were used, and their directions are shown in Table 1. HRP-conjugated secondary antibodies (1:5,000, GeneTex, Irvine, CA, USA) were also used. Other materials consisted of electrophoresis and membrane transfer device (Bio-Rad), RIPA lysate (Beyotime), protease inhibitor (cocktail) (Roche), ECL chemiluminescence substrate Kit (Biosharp, Hefei, China), defatted milk powder (Wondersun, Harbin, China), protein marker (Bio-Rad), BCA kit (Beyotime), and SDS-PAGE gel rapid preparation kit (Beyotime). Blotting was captured by Molecular Imager ChemiDocTM XSR+ Gel Imaging System (Bio-Rad) and analyzed using ImageJ software (NIH). In semiquantitative analysis of the target protein, each sample was normalized to β-actin.

Referring to the previous description^{48 (link)}, the protein from A549 and SCPA1 cells (5 × 10⁶) were obtained. The BCA method was used for protein quantitation. Then the proteins were separated by electrophoresis and transferred to PVDF membranes (Immobilon-P membrane, Millipore, Massachusetts, USA). After blocked with 5% (wt/vol) skimmed milk, the membranes were incubated with primary antibodies (shown in Table S1), and then analyzed by immune blotting of HRP-conjugated secondary antibodies (1:5000, GeneTex, USA). Blotting was visualized by an enhanced chemiluminescent chromogenic substrate (Biosharp, China), and captured by Molecular Imager ChemiDocTM XSR + Gel Imaging System (BIO-Rad, USA).