Goat anti rabbit igg hrp

The protocol of Western blot analysis was described previously (45 (link), 46 (link)). The antibodies used were as follows: anti-c-Met (Cell Signaling Technology, 8198S), anti-PD-L1 (Cell Signaling Technology, 13684S), anti-CD3ζ (Santa Cruz Biotechnology, sc-166275), anti-GAPDH (Proteintech, 60004-1- Ig), goat anti-mouse IgG-HRP (FDbio science, FDM007), and goat anti-rabbit IgG-HRP (FDbio science, FDR007). Final detection was performed with an enhanced chemiluminescence system (Tanon, China).

An equal amount of mitochondrial protein was subjected to 12% SDS–PAGE and then transferred onto a PVDF membrane (Millipore, Billerica, USA). The membranes were incubated at room temperature for 1 h in blocking buffer. After blocking, the PVDF membranes, which contained the proteins transferred from the gels, were cut into strips (~ 5 mm wide) according to the location (molecular weight) of the protein of interest. Each tailored PVDF membrane strip containing the target protein was then separately incubated over night at 4 ℃ with the corresponding primary antibody: STAT3 rabbit mAb (1:1000, Cell Signaling Technology, Boston, USA), phospho-STAT3 (Ser⁷²⁷) rabbit antibody (1:1000, Cell Signaling Technology, Boston, USA), VDAC rabbit mAb (1:1000, Cell Signaling Technology, Boston, USA) and actin mouse mAb (Bioss, Beijing, China). After washing, they were incubated with goat anti-rabbit IgG-HRP (1:5,000, FDbio, Hangzhou, China) or goat anti-mouse IgG-HRP (1:5,000, FDbio, Hangzhou, China). The immunoreactive bands were detected by ECL Substrate (FDbio, Hangzhou, China) and visualized with a GeneGnome XRQ chemiluminescence imager (Syngene, MD, UK). The grey values of the bands were obtained using ImageJ software.