Letm1

Radioimmunoprecipitation assay buffer (RIPA) buffer purchased from Beyotime Biotechnology (China) was used to extract total protein from tissues and cultured cells, and the supernatant was collected after centrifugation (12,000×g, 10 min, 4°C). After blocking with 5% skim milk, the membrane was incubated with primary antibodies against LETM1 (1:500, Santa Cruz, USA), AMPK (1:500, ABclonal, China), p-AMPK (1:500, ABclonal, China), Bcl-2 (1:500, ABclonal, China), p-Bcl-2 (1:500, Proteintech, USA), Bax (1:500, ABclonal, China), caspase-3 (1:500, Proteintech, USA), Beclin-1 (1:500, Proteintech, USA), p62 (1:500, Proteintech, China), LC3 (1:500, Proteintech, USA), and β-actin (1:2000, ABclonal, China) overnight. The membrane was then incubated with secondary antibodies (1:2,000, Proteintech, China) for 2 h the next day. The protein bands on the blot were analyzed.

Infected cells were harvested in ice-cold PBS, and proteins were solubilized in 9 M urea, 40 mM Tris HCl pH 7.4, and 0.15 M β-mercaptoethanol. Prior to immunoblotting, lysates were fractionated on 10% or 8% SDS polyacrylamide gels. Proteins were electrophoretically transferred to nitrocellulose membranes before incubation with antibodies at 4 °C overnight. The following antibodies were used in this study: HAdV-C5 E1A, M73 [54 (link)]; HAdV-C5 E1B-55K, 2A6 [55 (link)] or a rabbit antibody raised against GST-HAdV-C5 E1B-55K (produced in house); and p53 (DO-1; generous gift from David Lane). Commercial antibodies were obtained as follows: SQSTM1 (p62) (Abcam, Cambridge, UK); ATR, CSB, LETM1, MRE11, NBS1, TNKS 1 and 2, USP6, USP7, USP9, USP15, USP33, and USP34 (all from Santa Cruz Biotechnology, Dallas, TX, USA); and BLM, DNA ligase IV, and DNMT1 (all from Bethyl Laboratories, Montgomery, TX, USA).