Hrp conjugated rabbit mouse anti igg

The parental and transfected cells were washed with prechilled PBS and lysed in 1 × RIPA buffer (Sigma, St. Louis). The protein concentration was measured by the Bradford method with BSA (Sigma) as the standard. Equal amounts of protein extract (50 μg) were denatured with 8–10% SDS-PAGE, transferred to PVDF membranes (Millipore), and blocked with 5% non-fat milk in TBST (0.1 M, pH 7.4). Protein abundance of GAPDH (1:500; Abgent) served as a control for protein loading. Each sample was treated with rabbit polyclonal anti-ADAMTS5 (1:250; Abcam) or mouse monoclonal anti-IGFBP5 (1:250; Abcam) primary antibodies at 4 °C overnight. Membranes were incubated with secondary antibodies, HRP-conjugated rabbit/mouse anti-IgG (LI-COR Biosciences), diluted at 1:16,000 in TBST, for 2 h at room temperature. Protein bands were detected by the Odyssey infrared imaging system (LI-COR Biosciences).

For the ectopic expression of miRNAs, the protein was extracted at 48 h after transfection. Parental and transfected cells were washed with prechilled PBS and lysed in 1× RIPA buffer (Sigma). Then extraction and solubilization were performed. Equal amounts of protein extract (50 μg) were denatured with SDS/PAGE (10% gel). Protein abundance of GAPDH (1:500, Abgent) served as a control for protein loading. Each sample was treated with rabbit monoclonal anti-PTEN (phosphatase and tensin homolog deleted on chromosome ten) (1:500, Abcam), rabbit polyclonal anti-p-Akt (1:200, Abgent), rabbit polyclonal anti-Akt (1:200, Abgent), rabbit polyclonal anti-Bcl-2 (1:200, Abgent), rabbit polyclonal anti-BAX (1:200, Ax), rabbit polyclonal anti-caspase-3 (1:200, Proteintech), primary antibodies at 4°C overnight. Membranes were incubated with secondary antibody, HRP-conjugated rabbit/mouse anti-IgG (LI-COR Biosciences), diluted at 1:16000 in TBST, for 2 h at room temperature. Protein bands were detected by Odyssey infrared imaging system (LI-COR Biosciences).