Monoclonal anti α tubulin

Western blotting was performed as described previously [17 (link)], with modifications. Commercial polyclonal anti-SOD2 (Abcam, Cambridge, MA, USA) and polyclonal anti-NUDFV2 (Abcam) were used, and monoclonal anti-α-tubulin (Abcam) was used as a control. Briefly, the samples were washed by centrifugation in DPBS at 10,000 × g for 5 min. The pellets were re-suspended with lysis buffer containing 5% 2-mercaptoethanol and incubated for 10 min at RT. The samples were electrophoresed on a 12% SDS-polyacrylamide gel and transferred to polyvinylidene fluoride membranes (Amersham). The membranes were blocked with PBS-Tween containing 5% skim milk powder (blocking solution) for 3 h at RT. After washing, the membranes were incubated overnight with anti-NDUFV2 (1:3,000) and anti-SOD2 (1:5,000) diluted with blocking solution. Then, the membranes were incubated with horseradish peroxidase conjugated anti-rabbit immunoglobulin G (1:3,000, Abcam) for 1 h. After the membranes were washed, proteins were detected by enhanced chemiluminescence reagents. All bands were scanned with a GS-800 Calibrated Imaging Densitometer (Bio-Rad) and analyzed with Quantity One (v.4.6, Bio-Rad). The signal intensity ratios of the bands were calculated for SOD and NUDFV2/α-tubulin.

The immunoblot analysis was performed using an enhanced chemiluminescence (ECL) system (Millipore, Billerica, MA, USA), and results were monitored using the LAS-4000 imaging system (Fujifilm, Tokyo, Japan). Primary antibodies used in this study included polyclonal anti-RBM416 (link), monoclonal anti-PTBP2 (Abnova, Taipei, Taiwan), monoclonal anti-SRSF6 (EMD, Millipore), monoclonal anti-Nova1 (Abnova), monoclonal anti-actin (Millipore), monoclonal anti-α-tubulin (Abcam, Cambridge, UK), monoclonal anti-E-cadherin (Abcam), monoclonal anti-N-cadherin (Abcam), and monoclonal anti-FLAG M2 (Sigma-Aldrich, St. Louis, MO, USA). Signal intensities were evaluated using TotalLab Quant Software (where available?).