Rabbit anti p ulk 1

The cells were washed twice with ice-cold PBS and lysed in Cell Lysis Buffer (1x). Protein levels were determined using the Bradford method, and then the samples were mixed with Laemmli buffer and denatured at 95°C for 5 min. Equal amounts of proteins were separated on SDS/PAGE gels. All proteins were transferred to nitrocellulose membranes at 100 V. Membranes were washed for 5 min in TBS-Tween buffer (0.1% TBST) (100 mM Tris-buffered saline, 140 mM NaCl, and 0.1% Tween 20; pH 7.6) and the nonspecific bindings were blocked for 1 h at RT with 5% BSA in 0.1% TBST or with 5% nonfat milk solution in 0.1% TBST. Immunodetection was performed overnight at 4 °C using rabbit antiparkin (1:500; Cell Signaling), rabbit anti-AMPK (1:1000, Cell Signalling), rabbit anti-p-AMPK (1:1000, Cell Signalling), rabbit anti-Ulk-1 (1:200, Cell Signalling), rabbit anti-p-Ulk-1 (1:200, Cell Signalling), and antimouse total OXPHOS (1:500, Abcam) antibodies. Then, the membranes were washed three times (5 min) in TBST and incubated for 60 min at RT with antirabbit or antimouse secondary antibody (1∶4000) in a 5% nonfat milk/TBST. Antibodies were detected using chemiluminescent Clarity Western ECL Substrate (Bio-Rad Laboratories, Hercules, CA, USA) under standard conditions. Immunolabeling of GAPDH (rabbit anti-GAPDH; 1:40,000; Sigma-Aldrich) for cell lysates was performed as a loading control.

Cultured cells were washed three times with 4 °C PBS and then lysed with RIPA lysis buffer. The supernatant was centrifuged at 4 °C and 13800×g for 30 min to collect the protein. BCA Protein Assay Kit (Thermo, Life Technologies, California, USA) was used to determine the protein concentration. The samples were separated by SDS-polyacrylamide gel electrophoresis (PAGE) using 6–15% acrylamide gels and then transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). After incubation with primary and secondary antibodies, the protein bands were detected with chemiluminescence detection reagents (Millipore, Billerica, MA, USA). The following antibodies were used. Apoptosis Antibody Sampler Kit (#9930), Autophagy Antibody Sampler Kit (#4445), rabbit anti-BAX (#2772), rabbit anti-cytochrome c (#4272), rabbit anti-Parkin (#32833), rabbit anti-GAPDH (#2118), rabbit anti-ULK1 (#8054), rabbit anti-p-ULK1 (#5869), rabbit anti-AMPK (#5832) and rabbit anti-p-AMPK (#2535) were all from Cell Signaling Technology (CST, Beverly, MA, USA). Rabbit anti-PINK1 (ab23707), rabbit anti-mTOR (ab134903), rabbit anti-p-mTOR (ab109268) were from Abcam (Abcam, Cambridge, UK). Goat anti-rabbit IgG H&L (HRP) (ab205718) secondary antibodies were from Abcam (Abcam, Cambridge, UK). The integrated density of all the protein bands was analyzed with ImageJ software.