Amersham protran 0.45 nc nitrocellulose membranes

After appropriate treatment, cells were washed two times with PBS and lysed in RIPA buffer for 20 min in a 4 °C shaker. The lysates were collected by scraper and centrifuged at 14,000 RPM for 15 min. The protein concentrations of the cell extract were determined using a BCA Protein Assay Kit (Thermo Scientific, Rockford, IL, USA). Equal amounts of each protein samples (~30μg/lane) were separated by 10 or 15% SDS-polyacrylamide gel electrophoresis, and transferred onto Amersham Protran 0.45 NC nitrocellulose membranes (GE Healthcare Life Science, Seoul, Korea), and blocked with 5% milk in TrisHCl-buffered saline (TBS) containing 0.1% Tween 20 (TBST) for 1 h. Thereafter, the membranes were incubated at 4 °C overnight with various primary antibodies. After washing with TBST, the membranes were incubated with mouse and rabbit HRP-conjugated secondary antibodies at 23 °C and developed using ECL solution (LPS Solution, Daejeon, Korea), a chemiluminiscent detection method. The band intensities were quantified using Image J software (NIH, Bethesda, MD, USA) to analyze the difference in expression relative to the control.

Proteins were separated by SDS-PAGE on NuPage 4–12% Bis-Tris gels and transferred to Amersham Protran 0.45 NC nitrocellulose membranes (GE Healthcare) using wet transfer. Membranes were blocked using 5% w/v milk in Tris-buffered saline (TBS) with 0.1% Tween-20. Blots were probed using anti-VHL (CST-68547), anti-CRBN (Novus, NBP1-91810) and anti-β-tubulin hFAB-rhodamine (BioRad, 12004166) primary antibodies, followed by incubation with secondary anti-Rabbit IRDye 800CW (ab216773) or anti-rabbit HRP-conjugated (CST-7074) antibodies. Blots were developed using a Bio-Rad ChemiDoc MP Imaging System or the Amersham ECL Prime western blotting detection kit and Amersham Hyperfilm ECL film, as appropriate. Band quantification was performed using the ImageJ software. Band intensities were normalized to the β-tubulin loading control and reported as % of the average 0.1% DMSO vehicle intensity. Degradation data was plotted and analysed using Prism (Graphpad, version 7). DC₅₀ values (concentration to reach 50% maximal degradation) were estimated by fitting band intensity against log[concentration]. Apparent half-life values (time to reach 50% maximal degradation) were estimated by fitting band intensity against time using a single-phase exponential decay model.