Usp32 sc 374465

Cells were lysed in RIPA buffer, and protein concentration was measured using a BCA Assay Kit (Thermo Fisher Scientific, US). 20μg proteins were electrophoresed by SDS-polyacrylamide gels and transferred onto a nitrocellulose membrane (Millipore, US). The membrane was then blocked in non-fat milk for 1 h at room temperature and subsequently incubated with the primary antibodies overnight at 4°C. After washing with PBST (phosphate-buffered saline containing 0.05% Tween 20) for three or four times, then the membrane was incubated with the secondary antibody for 1 h at room temperature. Protein bands were visualized using the Odyssey Infrared Imaging System (Li-COR Biosciences). The antibodies used in this study were as follows: USP32 (sc-374465, Santa Cruz Biotechnology, 1:100), β-actin (#81178, Santa Cruz Biotechnology, 1:1,000), SMAD2 (#12570-1-AP, Proteintech, Wuhan, China, 1:500), p-SMAD2 (#18338, Cell Signaling Technology, US), and FLAG (F1804, Sigma-Aldrich, US).

To screen functional DUBs that regulate SLC35F2 protein, we co-transfected HeLa cells with sgRNA targeting 50 DUBs and Cas9 plasmids. The cells were washed with phosphate-buffered saline (PBS) (PAN Biotech, Aidenbach, Germany), harvested, lysed with a lysis buffer, and incubated on ice for 20 min. The samples were then centrifuged and the resulting supernatants collected and kept in separate tubes. A Bradford assay (Biorad, CA, USA) was used to estimate protein concentrations. Western blots were performed using SLC35F2-specific antibodies (25526-1-AP; 1:1000) from Proteintech (IL, USA). The antibodies used in our experiments were as follows: γH2AX (05-636; 1:1,000) from Millipore (MA, USA); SLC35F2 (NBP1-59890; 1:1,000) from Novus (CO, USA); USP19 (25768-1-AP; 1:1000) and USP49 (18066-1-AP; 1:1,000) from Proteintech; H2AX (sc-517336; 1:1000), SLC35F3 (sc-515378; 1:1000), USP32 (sc-374465; 1:1,000), GAPDH (sc-47724; 1:1,000), Myc (sc-40; 1:1,000), and Calnexin (sc-23954, 1:100) from Santa Cruz Biotechnology (TX, USA); and Flag (M185-3L; 1:1,000) from MBL International (MA, USA), and AKT (9272, 1:1000), p-AKT (9271, 1:1000), PI3K (4292, 1:1000), p-PI3K (4228, 1:1000), mTOR (2983, 1:1000), p-mTOR (2971, 1:1000) and Survivin (2808, 1:1000) were procured from Cell Signaling Technology (MA, USA).