Phospho psmad2

Melanoma cells were cultured in six-well dishes then transfected with indicated siRNA. 72 h after transfection, cells were lysed in buffer containing 50 mmol/L Tris-HCl (pH 7.4), 150 mmol/L NaCl, 1% Triton X-100, protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific). Samples were dosed using Pierce BCA Protein Assay kit (ThermoFisher) and 30 µg of total extracts were resolved by 10% SDS-PAGE, transferred to nitrocellulose membranes, and then exposed to the appropriate antibodies: anti-βactin-HRP (Sigma-Aldrich A3854, diluted at 1: 5000), Vinculin (Cell Signaling Technology 13901 S, diluted at 1:1000), TFEB (Cell Signaling Technology 4240 S, diluted at 1:1000), LC3A/B (Cell Signaling Technology 4108, diluted at 1:1000), IGF2R (Cell Signaling Technology 14364 S, diluted at 1:1000), Cathepsin B (Cell Signaling Technology 31718, diluted at 1:1000), TFE3 (Cell Signaling Technology 14779 S, diluted at 1:1000), Phospho-pSMAD2 (Cell Signaling Technology 18338, diluted at 1:1000), SMAD2 (Cell Signaling Technology 5339, diluted at 1:1000), laminA/C (Cell Signaling Technology 2032, diluted at 1:1000), Phospho-4E-BP1 (Ser65) (Cell Signaling Technology 9451, diluted at 1:1000), 4E-BP1 (Cell Signaling Technology 9644, diluted at 1:1000).
Proteins were visualized with the ECL system (GE Healthcare) using horseradish peroxidase conjugated secondary antibodies.

Cells were lysed in RIPA Lysis Buffer and phenylmethylsulfonyl fluoride (PMSF) (Thermo Fisher Scientific, Inc.) and the lysates were centrifuged at 10,000 × g for 15 min at 4°C according to the manufacturer's protocols. Protein concentration was determined using Bradford reagent (Sigma-Aldrich; Merck KGaA). Proteins (15 µg) were separated by 10% SDS-PAGE and transferred onto nitrocellulose membranes (EMD Millipore). Membranes were blocked with 5% fat-free milk for 1 h at room temperature and incubated with primary antibodies against BUB1 antibody (cat. no. ab195268; 1:1,000; Abcam), anti-SMAD2 (cat. no. 5339; 1:1,000; Cell Signaling Technology, Inc.), phospho- (p) SMAD2 (cat. no. 3104; 1:1,000; Cell Signaling Technology, Inc.), PCNA (cat. no. 13110; 1:1,000 dilution; Cell Signaling Technology, Inc.), Ki67 (cat. no. 2586; 1:1,000; Cell Signaling Technology, Inc.), Flag (cat. no. 8164; 1:1,000; Cell Signaling Technology, Inc.) and GAPDH (cat. no. 5174; 1:1,000; Cell Signaling Technology, Inc.) at 4°C overnight. Membranes were then incubated with anti-rabbit horseradish peroxidase-conjugated secondary antibody (cat. no. 7074; 1:5,000; Cell Signaling Technology, Inc.) for 2 h at room temperature. The immunoreactive protein bands were visualized using an enhanced chemiluminescence kit (Pierce; Thermo Fisher Scientific, Inc.) and a Gel Dox XR system (Bio-Rad Laboratories, Inc.).