Horseradish peroxidase conjugated goat anti mouse antibody

Tissue samples and tenocytes were lysed by RIPA (Beyotime, Shanghai, China). Western blotting was performed based on standard protocols. Antibodies used in this research were shown here: anti-cleaved caspase 3 (1:1500, Cell Signaling Technology, Danvers, MA), anti-Bax (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA), anti-Bcl-2 (1:1200, Santa Cruz Biotechnology), anti-phospho-AKT (1:1000, Cell Signaling Technology), anti-IGF1R (1: 1000, Cell Signaling Technology), anti-AKT (1: 1000, Cell Signaling Technology), anti-ERK1/2 (1: 1000, Cell Signaling Technology), anti-phospho-ERK1/2 (1:800, Cell Signaling Technology), anti-p-IGF1R (Tyr1135) (1:600, Cell Signaling Technology), anti-p-IGF1R (Tyr980) (1:800, Cell Signaling Technology), and anti-GAPDH (1:2000, Abcam, Cambridge, MA). Horseradish peroxidase-conjugated goat anti-rabbit antibody (Cell Signaling Technology) and horseradish peroxidase-conjugated goat anti-mouse antibody (Cell Signaling Technology) were used as the secondary antibody. Experiments were independently performed for at least 3 times.

Astrocytes were infected with MSCV-Mash1 and MSCV retroviruses for 7 days, following which the protein expression of β-tubulin was analyzed. Infected cells were lysed with lysis buffer (P0013B; Beyotime, Shanghai, China) containing phenylmethyl sulfonylfluoride. Lysates were centrifuged at 12,000 × g at 4°C for 10 minutes. Protein concentrations were determined using BCA Protein Assay Kit (Cat. No. 23227; Pierce, Rockford, IL, USA). Proteins were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane using a standard protocol. The membrane was incubated with the mouse primary antibody β-tubulin (1:1,000) and with secondary horseradish peroxidase-conjugated goat anti-mouse antibody (1:20,000; Cell Signaling Technology, Boston, MA, USA). The labeled proteins were visualized by chemiluminescence using enhanced chemiluminescence detection kit (Cat. No. 32106; Pierce).