Anti mgmt antibodies

Co-immunoprecipitation (Co-IP) analyses were conducted according to a previous report [26 (link)]. To exclude the contaminating effect of DNA attached to tested proteins, 20 U/ml of DNase I (Roche) was added in lysis buffer. In brief, the cell lysates were subsequently sonicated, washed, and incubated with anti-MGMT antibodies (Abcam) or negative control IgGs (Santa Cruz Biotechnology). After incubation for 24 h, Pierce Protein A/G UltraLink Resin (Thermo Fisher Scientific) was added to capture immune complexes. After washing, the precipitated proteins were resuspended in nonreducing loading buffer and heated at 95 °C for 5 min before Western blot analyses.

Tumor cells were cultured on coverslips. After the cells attached to the slips over 24 hours, FM19G11 was added to the culture media for 72 hours. The cells were then fixed with 4% formaldehyde (10 minutes), permeabilized with 0.1% Triton X‐100 for 5 minutes, and blocked with 1% BSA/10% normal goat serum for 1 hour. The cells were then incubated with anti‐MGMT antibodies (Abcam, Cambridge, USA) overnight at 4°C in the dark, followed by incubation at room temperature for 1 hour with secondary antibodies (Abcam). Then, the slips were rinsed in PBST once and in PBS twice, and mounted with DAPI mounting solution. The slips were immediately examined under a fluorescence microscope.