Horseradish peroxidase conjugated immunoglobulin g igg

Western blot analysis was performed as previously reported^{52 (link)}. Briefly, the cells were lysed with cell lysis buffer (Cell Signaling Technology, Danvers, MA, USA), the proteins were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and then electrophoretically transferred to Immobilon-P membranes (Millipore, Billerica, MA, USA). Then, the membranes were probed with anti-ERK1/2 polyclonal antibodies (1:1000, Cell Signaling Technology), anti-phospho ERK1/2 (Thr202/Tyr204) monoclonal antibodies (1:2000, BD Biosciences), anti-Mfn1 monoclonal antibodies (1:1000, Cell Signaling Technology), and anti-β-actin monoclonal antibodies (1:5000, Sigma-Aldrich). The membranes were then incubated with secondary antibodies against rabbit or mouse horseradish peroxidase-conjugated immunoglobulin G (IgG, Cell Signaling Technology). The bands were visualized using an enhanced chemiluminescence (ECL) western blotting analysis system (GE Healthcare, Buckinghamshire, UK) and the images were acquired using an LAS-3000 imager (FUJIFILM UK Ltd., Systems, Bedford, UK).

Cells were seeded into 60 mm cell culture plates at a density of 4 × 10⁵ for about 12~16 h. The cells were lysed using RIPA lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China) containing protease inhibitor cocktail and phenylmethylsulfonyl fluoride (PMSF). The lyses was centrifuged at 1.5 × 10⁴ cells/well for 15 min at 4 °C and the concentration of these supernatants were assayed by BCA Protein assay kit (Beyotime Institute of Biotechnology, Shanghai, China). To separate these proteins, 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used, and they were then transferred into nitrocellulose (NC) membranes (PALL, Stevenage, UK). Then, the NC membranes were incubated with specific primary antibodies, including Ki67 (ABclonal Technology, Wuhan, China), STAT3 (ABclonal Technology, Wuhan, China), p-STAT3(Cell Signaling Technology, MA, USA), EXOSC5, GAPDH (Cell Signaling Technology, MA, USA) at 1:1000 overnight at 4 °C. In addition, horseradish peroxidase-conjugated immunoglobulin G (IgG) (Cell Signaling Technology, MA, USA) was used to incubate the membranes at 1:5000 for 1 h at room temperature. Immunoreactive protein bands were detected with an Enhanced Chemiluminescence (ECL) Detection Kit (Amersham, Piscataway, NJ, USA). Image J software was used to analyze the relative levels of proteins normalized to the expression of GAPDH.