S2 cells were homogenized in a RIPA lysis buffer [5 mmol/L Tris–HCl, pH 8.0, 150 mmol/L NaCl, 1% (v/v) IGRPA CA-630, 0.5% (w/v) sodium deoxycholate and protease inhibitor cocktail (Roche)] and centrifuged at 12,000×g for 10 min at 4 °C to pellet nuclei and cell debris. The supernatant was collected and the protein concentration was assayed using a BCA protein assay kit (Beyotime). 20 μg of the protein samples were subjected to 12% sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF (polyvinylidene fluoride) membranes. The blotted membranes were immunoblotted with primary antibodies: rabbit anti-Prominin-like (1:2000), mouse anti-Tubulin (1:1000, DHSB E7), mouse anti-VDAC (1:1000, Abcam), mouse anti-Cytochrome C antibody (1:1000, Abcam 13575), mouse anti-ATP5a (1:1000, Abcam), mouse anti-COX IV (1:1000, Abcam), Rabbit anti-CG9172 (ND20) (1:500, Abgent), rabbit anti-HA (1:1000, Abcam), mouse anti-Flag (1:1000, Cell Signaling), rabbit anti-LAMP-1 (1:1000, Abcam). After secondary anti-rabbit horseradish peroxidase antibody labelling (Pierce; 1:5000), ChemiLucent™ ECL detection reagents (Millipore) was used to detect proteins and images were taken using the Chemiluminescence Imaging System (Clinx Science Instruments) as previously reported [34 (link)].
Free full text: Click here
