Usp37

Cellular extracts were generated in EBC lysis buffer (50 mM Tris (pH8.0), 120 mM NaCl, 0.5% Nonidet P-40, 1 mM DTT, and protease and phosphatase inhibitor tablets) (Thermo Fisher USA). Protein concentration was quantified by Pierce BCA assay (Thermo Fisher, USA), and samples were prepared by boiling them in Laemmli buffer for 5 min. Equal amounts of whole cell lysates were resolved by hand-cast SDS-PAGE and transferred to PVDF membranes (Millipore). All blocking and primary antibody steps were performed in 5% BSA diluted in TBST (137 mM NaCl, 2.7 mM KCl, 25 mM Tris pH 7.4, 1% Tween-20). All primary antibody incubations were performed with shaking at 4℃ overnight. After the transfer of the separated proteins to PVDF membranes, the membranes were probed with the following primary antibodies: USP37 (dilution 1:1000; Elabsciences), β -actin (dilution 1:10,000; Sigma, MO, USA), γ-H2AX (at dilution 1:1000; from Santa Cruz). After overnight incubation, membranes were extensively washed and then incubated with either secondary goat anti-mouse/rabbit HRP-conjugated antibodies (Cell signalling technology). Separated protein bands were visualized using chemiluminescence HRP substrate (Bio-Rad, CA, USA), and the membranes were imaged and analyzed using the Chemi Doc™ imaging system from Bio-Rad (CA, USA).