Figure 6
The protein expression levels of Bax, Bcl-2, LC3B, and P62 was detected by western blot analysis. SW620 cells were seeded into a 6-well plate at a density of 5 × 105 cells per well, cultured overnight and treated with or without Cu-Cy-PDT. The cells were lysed for 30 minutes in 1 × RIPA buffer that contained protease and phosphatase inhibitors. Samples containing equal amounts of protein (25 μg) were resolved on SDS-PAGE in a 10–15% gel and then transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA, USA). The membrane was then blocked in 5% non-fat milk for 2 h at room temperature, incubated with primary antibody Bax, Bcl-2, ATG7, LC3B, and Actin antibody (Cell Signaling, Danvers, MA, USA) overnight at 4 °C, washed with TBST, and incubated with a secondary antibody for 2 h at room temperature. The immunoblots of the incubated membranes were visualized with an enhanced chemiluminescence (ECL) Kit (CW Bio).
