Anti map1lc3a antibody

The CHK2 inhibitor BML-277 was purchased from Merck and Millipore (USA). Rabbit polyclonal anti-CHK2 antibody, rabbit monoclonal anti-γ-H2A.X antibody and anti-MAP1LC3A antibody were purchased from Abcam (Cambridge, UK). Anti-α-tubulin-FITC antibody and Hoechst 33342 were purchased from Sigma (St. Louis, MO, USA). Alexa Fluor 488 goat anti-rabbit antibody and Alexa Fluor 594 goat anti-rabbit antibody were purchased from Invitrogen (Carlsbad, CA, USA).

After treatment with the indicated condition, cells were harvested and washed with cold phosphate-buffered saline (PBS) and followed by incubation in Cell Lysis Buffer (Beyotime Biotechnology, China) in ice for at least 20 min. The lysates were centrifuged at 12,000 ×g for 10 min, and the supernatants were collected. Equal amount of protein was run on SDS-PAGE gels and subsequently electro-transferred to polyvinylidene fluoride (PVDF) membranes. The membrane was washed 3 times with TBST. Then, the primary antibodies were diluted with the Western antibody dilutions and incubated at 4°C in a horizontal shaker overnight, dilution ratio: Anti-MAP1LC3A antibody (1 : 2500; Abcam, Cambridge, England, UK) and Anti-mTOR antibody (1 : 1000; Abcam). The secondary antibody was diluted with 5% skim milk powder and incubated at room temperature for 60 min, ImmunoPure goat anti-rabbit IgG antibody (H+L) (1 : 5000; Thermo-Fisher, Waltham, MA, USA). Then we lifted the PVDF membrane and drained the excess detergent solution. The film was wrapped with a clean plastic wrap and placed in the development folder. ECL visualization was performed, and the gel images were analyzed with Image J (NIH image, Bethesda, MD, USA).