Ab106926

Cells received siRNA transfection were incubated with 50 μL cell lysis solution for 30 minutes, then transferred into new tubes and centrifuged at 16,000 rpm at 4°C for 15 minutes. The amount of protein in the supernatant was determined by BCA method (BCA Protein Detection Kit, Google Bio). After quantification, samples were mixed with sodium dodecyl sulfate (SDS) loading buffer and separated by SDS-polyacrylamide gel electrophoresis (Western Blotting Kit, Google Bio). Western transfer was performed using the iBlot system (Invitrogen). PVDF membranes (Millipore, Billerica, MA) were blocked for 1 hour in Tris-buffered saline (pH 7.4) with 1% Tween-20 (TBS-T) with 5% milk and incubated in primary antibody overnight at 4°C. Following washed about 10 minutes in TBS-T for three times, the membrane was incubated with secondary antibodies for 1 hour at 37°C. The membrane was washed again in TBS-T thrice for 10 minutes prior to visualization using enhanced chemiluminescence (ECL, Thermo Fisher Scientific, Waltham, MA). Antibody for IREB2 (ab10-6926, Abcam, Cambridge, MA) were used at 1:1,000 dilution, and detected using a goat mouse horseradish peroxidase conjugate secondary antibody (Santa Cruz Biotechnology) at 1:5,000 dilution.