Goat anti rabbit or anti mouse secondary antibody

Proteins were retrieved from cells and equal quantities of proteins were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose membrane (Millipore, Bedford, MA, USA), and blocked with 5% bovine serum albumin. Then the membrane was incubated with MYC (Abcam, Hong Kong, China) or β-actin (Abcam) specific primary antibodies. After being washed by TBST for three times, the membrane was incubated with goat anti-rabbit or anti-mouse secondary antibody (Abcam) and visualized with enhanced chemiluminescence.

Cytoplasmic and nuclear proteins were extracted from cells using BeyoECL Plus Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime) supplemented with protease inhibitors and phosphatase inhibitors. Western blotting was performed as previously described [11 (link)]. Primary antibodies specific to GAPDH (1:1000 dilution, Cell Signaling Technology, Danvers, Massachusetts, USA), H3 (1:1000 dilution, Cell Signaling Technology), and APC (1:1000 dilution, Abcam, Cambridge, UK) were used. The blots were then incubated with goat anti-rabbit or anti-mouse secondary antibody (1:3000 dilution, Abcam) and visualized by Immobilon Western Chemiluminescent HRP Substrate (Millipore, St. Louis, MO, USA).

The expression levels of involved proteins were measured by western blotting analysis and analyzed by ImageJ software. Briefly, the cell samples at 24 h after BL-PDT were collected and lysed in 1 × radioimmunoprecipitation assay buffer with 1% phenylmethylsulfonyl fluoride for 20 min on ice. After quantification by a bicinchoninic acid assay kit (Beyotime, Shanghai, China), lysates were boiled for 10 min at 100 °C, and aliquots of 20 μg of protein were separated by 10% or 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes that were then blocked with 5% skim milk for 1 h at room temperature and incubated with primary antibodies on a shaker (4 °C, overnight). After 3 times of washing with TBST and 2 h of incubation with the corresponding secondary antibody at room temperature, the immunoblots on the membranes were observed using an enhanced chemiluminescence detection kit (Millipore, Burlington, MA, USA). The primary antibodies included LC3 and PARP antibodies (all from Cell Signaling Technology, USA); p62, Beclin-1, Bax, Bcl-2, Caspase 3, and Caspase 9 antibodies (all from Abcam, UK); and PI3K, p-PI3K, Akt, p-Akt, mTOR, and p-mTOR antibodies (all from Abmart, China). Mouse anti-human β-Actin antibody and goat anti-mouse or anti-rabbit secondary antibody were purchased from Abcam.