Polyvinylidene fluoride membrane

DROSHA KO cell lines were lysed in radioimmunoprecipitation assay (RIPA) lysis buffer (Thermo Scientific, Rockford, IL, USA), with protease inhibitor (Roche, Mannheim, Germany). Samples were mixed suspended in 2× Laemmli sample buffer supplemented with beta-mercaptoethanol. The mixtures were denatured by boiling and loaded onto a 10% SDS-polyacrylamide gel. Electrophoresis was performed to separate each sample according to size and subsequently transferred to a polyvinylidene fluoride membrane (Millipore, Billerica, MA, USA). Following blocking in PBS with 0.1% Tween 20 and 5% nonfat dry milk, the polyvinylidene fluoride membrane was incubated with a monoclonal anti-DROSHA rabbit antibody (Abcam, Cambridge, MA, USA) or mouse anti-β-actin (Sigma-Aldrich, St. Louis, MO, USA). Anti-rabbit and anti-mouse IgG HRP-linked secondary antibody (Cell Signaling, Danvers, MA, USA) was used for detection by chemiluminescence by SuperSignal West Dura Extended Duration Substrate (Thermo Scientific, Rockford, IL, USA).

Total protein samples were extracted from neurons (2 × 10⁶ cells per well, six‐well plates) with the corresponding treatment using cell lysis buffer for western blot analysis (Solarbio). The concentration of the proteins was measured using a bicinchoninic acid assay. After boiling for 10 min, the samples were separated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (12% polyacrylamide gels), transferred to polyvinylidene fluoride membranes (Solarbio), and blocked with 5% nonfat milk for 2 h at room temperature. The polyvinylidene fluoride membranes were then incubated with the following primary antibodies: Bcl‐2 (1:1000; Abcam), cleaved caspase‐3, Bax, and β‐actin (1:1000; all purchased from Cell Signaling Technology) at 4°C for 12 h. The membranes were washed three times with Tris‐buffered saline containing Tween 20 (TBST; Solarbio) and then incubated with a secondary antibody anti‐rabbit immunoglobulin G (IgG) (H+L) (1:2000; Cell Signaling Technology) for 1 h at room temperature. After washing with TBST three times, the signals were visualized using a chemiluminescent imaging system (GelView6000M; BLT). The relative expression of each target protein was determined by Quantity One software 4.6.2 (Bio‐Rad) and normalized to that of β‐actin. Experiments were repeated six times, and the average was calculated.