Immunoblotting of Nuclear Proteins

Cell lysates were prepared with 0.5% NP-40 buffer (50 mM Tris–HCl [pH 7.5], 0.5% Nonidet P-40, 150 mM NaCl, 50 mM NaF, 1 mM dithiothreitol, 1 mM NaVO₃, 1 mM PMSF, 2 µg/ml aprotinin, 2 µg/ml leupeptin, 10 µg/ml trypsin inhibitor, and 150 µg/ml benzamidine) and subjected to immunoblotting, as previously described [15 (link), 16 (link)]. Benzonase nuclease (125 U/ml; Millipore, MA, USA) and 2 mM MgCl₂ were supplemented for detection of nuclear proteins. The relative protein expression levels of Trop-2 were measured using a densitometer and normalized to tubulin. The following antibodies were used: rabbit monoclonal antibody against Trop-2 (Abcam, Cambridge, UK), AKT (Cell Signaling Tech, MA, USA), SQSTM1/p62 (D5E2; Cell Signaling Tech), rabbit polyclonal antibody against ERα (Millipore), BRCA1 (C20; Santa Cruz, CA, USA), HER2 (Cell Signaling Tech), pAKT (phospho-Ser473 AKT; Cell Signaling Tech), TFEB (Cell Signaling Tech), pRSK (Pan phospho-Ser221, Ser227, Ser218, Ser232; Invitrogen), LC3 (2775; Cell Signaling Tech), mouse monoclonal antibody against γH2AX (JBW301, Millipore), RSK1 (A-10; Santa Cruz), RSK2 (E-1; Santa Cruz), β-actin (6276; Abcam), and α-tubulin (DM1A; Richard-Allan Scientific, MI, USA).