Odyssey clx 2

Protein samples from lung tissues, A549 and BEAS2B cells were analyzed (21 (link)). Protein samples (70 µg) were extracted with lysis buffer (Beyotime Institute of Biotechnology) supplemented with 1% protease inhibitor solution. The concentration of the proteins was determined using a BCA Protein Assay kit (Beyotime Institute of Biotechnology). Subsequently, proteins (20 µg) were separated by 10% SDS-PAGE and transferred to nitrocellulose membranes. The membranes were blocked in PBS containing 5% non-fat milk for 2 h at room temperature and incubated at 4°C overnight with the following primary antibodies: SHCBP1 (1:1,000; cat. no. ab184467; Abcam), PTEN (1:500; cat. no. ab32199; Abcam) and GAPDH (1:2,000; cat. no. TA802519; OriGene Technologies, Inc.). Following washing, the membrane was incubated with secondary anti-rabbit immunoglobulin G (IgG; 1:1,000; cat. no. A3687; Sigma-Aldrich; Merck KGaA) and anti-mouse IgG (1:1,000; cat. no. M8770; Sigma-Aldrich; Merck KGaA) antibodies at room temperature for 1 h. Images were captured on an Odyssey CLx Infrared Imaging system (LI-COR Biosciences). Subsequently, the blots were semi-quantified using Odyssey CLx 2.1 software (LI-COR Biosciences). Data were normalized to GAPDH as an internal control.