Supersignal r west femto maximum sensitivity substrate

Cells were harvested and homogenized for total protein extraction. Equivalent amounts of protein extracts were separated by SDS-PAGE and then transferred to nitrocellulose membranes (Amersham Biosciences, Buckinghamshire, UK). Rabbit anti-CCT2 (1∶1000; ProteinTech Group Inc, Wuhan, China), anti-stathmin(1∶500; ProteinTech Group Inc), and anti-β-actin antibody (1∶2000; Cell Signaling Technology, Beverly, MA, USA) were used as the primary antibodies and horseradish peroxidase conjugated goat anti-rabbit IgG (Thermo Scientific Pierce, Rockford, IL, USA) was used as the secondary antibody. Immunoreactive bands were detected using SuperSignal (R) West Femto Maximum Sensitivity Substrate (Thermo Scientific Pierce). The analysis was performed three times.

Western blot analysis was performed as described previously [13 (link), 14 (link)], with primary antibodies including the following: rabbit anti-PDIA6 (1 : 1,500; Abcam, Cambridge, MA, USA), anti-beta-centractin (1 : 500; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), and anti-β-actin antibody (1 : 2,000; Cell Signaling Technology, Beverly, MA, USA). HRP-conjugated secondary antibody (Santa Cruz Biotechnology) was used as the secondary antibody. The protein signals were detected using SuperSignal (R) West Femto Maximum Sensitivity Substrate (Thermo Scientific Pierce). The grayscale value quantified using ImageJ software was used to calculate relative protein expression. The assays were independently repeated at least three times.