Anti pdcd4

Cells were lysed in radio immunoprecipitation assay (RIPA) buffer (Yeasen, Shanghai, China), and protein was collected by centrifugation (4°C, 12,000 × g, 10 min). Protein concentrations were determined by bicinchoninic acid (BCA) protein assay kit (Yeasen, Shanghai, China). Proteins were separated by 15% SDS-PAGE and subsequently transferred onto polyvinylidene fluoride (PVDF) membranes. Membranes were blocked with 5% non-fat milk in Tris-buffered saline Tween 20 (TBST) for 2 h with gentle shaking at room temperature, then incubated overnight with the following primary antibodies: anti-LC3B (1:1,000; Abcam, Cambridge, MA, USA), anti-SQSTM1 (1:1,000; Abcam, Cambridge, MA, USA), anti-PDCD4 (1:1,000; Abcam, Cambridge, MA, USA), and anti-β-tubulin (1:6,000; Abcam, Cambridge, MA, USA). The membranes were washed three times with TBST for 10 min and incubated for 1 h with secondary antibodies (1:2,000, Proteintech, Rosemont, IL, USA) at room temperature. Protein bands were scanned using a Tanon 4600SF (Tanon, Shanghai, China). The density of protein bands was quantified with Image-Pro Plus 6.0 software, and the ratio of target protein to β-tubulin, which reflects the changes in expression levels, was calculated.