Secondary horseradish peroxidase hrp conjugated antibodies

For total protein extraction, iLIN28B and CTRL cells were trypsinized (PAN Biotech, Aidenbach, Germany), washed with cold PBS once, and resuspended in cold lysis buffer (Biosource International, Camarillo, CA, USA) supplemented with protease and phosphatase inhibitors (Sigma-Aldrich). Immunoblotting was performed with 20 µg of the proteins quantified with Pierce BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer’s recommendations. Proteins were loaded on 4–20% gradient gel and SDS-PAGE (Bio-Rad, Hercules, CA, USA) was performed as described elsewhere [36 (link)]. Primary antibodies used in the study were anti-LIN28B (Cell Signaling, Danvers, CA, USA; 4196S) and anti-Vinculin (Santa Cruz, Dallas, TX, USA; sc-73264). For protein visualization, secondary horseradish-peroxidase (HRP)–conjugated antibodies (Sigma-Aldrich) were used. Enhanced chemiluminescence (ECL) Western blotting detection reagents (ECL^TM Select, Merck Life Science S.r.l., Milan, Italy) were used for protein band acquisition with the iBright Imaging Systems (Life Technologies, Thermo Fisher Scientific, Waltham, MA, USA).

Cells were lysed using the CHAPS buffer, and the protein concentrations were measured using the Bradford assay kit (Beyotime). An equal amount of protein (20 μg) was resolved on 10% SDS‐PAGE and transferred to polyvinylidene fluoride (PVDF) membranes. Next, the PVDF membranes were blocked with skimmed milk for 1 h at room temperature, following which the membranes were incubated with primary antibodies overnight at 4°C. Afterward, the membranes were incubated with secondary horseradish peroxidase (HRP)‐conjugated antibodies (Sigma‐Aldrich). Immunoprecipitation was performed as described previously to detect the activation of Bax.⁸ The following primary antibodies were used: Caspase‐3 (CST, USA), Bcl‐2 (CST), Smac/DIABLO (CST), Cytochrome c (CST), Bax (CST), Bax (6A7) (CST), GRP78 (Abcam), CHOP (Abcam), phospho‐JAK2 (Abcam), phospho‐STAT3 (Abcam), and GAPDH (Sigma‐Aldrich). Secondary antibodies were obtained from Sigma‐Aldrich.