Anti rabbit egfr

Immunocytochemical analysis was carried out as previously described (Cheng et al., 2017 (link)). Antibodies used include anti-mouse A2B5 IgM, anti-BrdU IgM, O4 IgM (1:1 dilution in DPBS + 10% goat serum), anti-mouse Olig2 (1:1000, Millipore), anti-mouse MBP (1:500, Abcam), anti- rabbit EGFR (1:200, Abcam), anti-mouse GFAP (1:300, Chemicon), anti-rabbit Nestin (1:2000, Covance), and anti-rabbit neurofilament (1:100, Sigma). The Alexa-488 or Alexa-594 conjugated secondary antibodies were obtained from Molecular Probes (Thermo fisher). The nucleic acid dye 4′,6-diamidino-2-phenylindole (DAPI) was obtained from Roche.

The primary antibodies used for immunoblot analysis and immunohistochemical staining were rabbit anti-EGFR, mouse anti-vinculin, mouse anti-Tom23, goat anti-Tim23, rabbit anti-Mfn1, mouse anti-Drp1 (all above from Santa Cruz Biotechnology, CA, USA), mouse anti-MTCO1 (Abcam, Cambridge, UK), rabbit anti-phospho-EGFR (Tyr1068), rabbit anti- phospho-Drp1 (Ser637) (all above from Cell Signaling Technology, Inc., MA, USA), mouse anti-OPA1 (BD Biosciences, NJ, USA) and mouse anti-myc, mouse anti-Flag, mouse anti-β actin antibody (all above from Sigma-Aldrich, Inc., MO, USA). The primary antibodies used for immnofluorescence staining were rabbit anti-EGFR (Abcam, Cambridge, UK) antibody.

Cells were plated on coverslips for 24 h incubation, and were fixed in 4% PFA for 30 min at room temperature. Samples were incubated with primary antibodies overnight at 4°C, followed by Alexa Fluor^® 488-conjugated or Alexa Fluor^® 594-conjugated second antibodies (1:500, Jackson Immunoresearch Laboratories, West Grove, PA, United States) for 2 h at room temperature. The primary antibodies used for incubation are as follows: rabbit anti-Sox2 (1:400, Abcam), goat anti-GFAP (1:400, Abcam), rabbit anti-Doublecortin (DCX) (1:400, Abcam), rabbit anti-EGFR (1:400, Abcam), rabbit anti-Map2 (1:800, Sigma-Aldrich), rabbit anti-Oligodendrocyte Marker O4 (1:100, Sigma-Aldrich). The stained sections were imaged by a fluorescence microscope (Eclipse Ti, Nikon).

Tissue or cell samples were lysed in RIPA lysis buffer (Applygen, Beijing, China) containing phenylmethylsulfonyl fluoride (PMSF, Beyotime, Shanghai, China). Equal amounts of protein, which were determined by a BCA Protein Assay Kit (Thermo Pierce, Waltham, USA), were separated via 12% Bis-Tris NUPAGE gradient gels with MOPS running buffer and then transferred onto polyvinylidene difluoride (PVDF) membranes (Thermo Fisher, Waltham, USA). After blocking with 5% skim milk in Tris-buffered saline (TBS) for 1 h, the membranes were blotted with primary antibodies on a shaker overnight at 4 °C. Primary antibodies were as follows: mouse anti-Rac1 (1:1000, Thermo), rabbit anti-α-SMA (1:1000, CST), rabbit anti-EGFR (1:1000, Abcam), rabbit anti-EGFR (phosphor Y1068) (1:1000, Abcam), and rabbit anti-GAPDH (1:1000, CST). The membranes were washed three times using TBS with 0.05% Tween-20 for 10 min each, followed by incubation with corresponding secondary horseradish peroxidase (HRP)-conjugated antibodies (1:5000, Lianke Technology) for 1 h at room temperature. Blots were developed using an enhanced chemiluminescence kit (Biological Industries) and visualized via X-ray films. The levels of protein expression were quantified by ImageJ software.