Anti gstp1

Western blot was performed according to a standard method. The total protein was extracted with RIPA buffer (Beyotime), according to the manufacturer's instructions. The concentration was determined by the BCA method. Twenty-five micrograms of protein was used to run on a 10% polyacrylamide (Beyotime) gel and transferred to a PVDF membrane underneath. The membrane was blocked in 5% non-fat milk (Bio-Rad). Primary antibodies and secondary antibodies (Proteintech) were used according to manufacturer's protocol. ECL method was used and the blot was developed under the gel electrophoresis imager (Bio-Rad). The following primary antibodies were used: anti–Gstp1 (Abcam), anti–Mek1 (upstate), anti–p-Mek1/2 (Cell Signaling Technology), and anti–GAPDH (Proteintech).

Immunoblotting was performed after 10% or 12% SDS-PAGE by probing with primary antibodies at the indicated dilutions: anti-GAPDH (1: 1000, mouse monoclonal; Milipore, Billerica, MA, USA), anti-COL6A3 (1:200, mouse monoclonal; Santa Cruz Biotech, Santa Cruz, CA, USA), anti-GSTP1 (1:1000, rabbit polyclonal; Abcam, Cambridge, UK), anti-FLNA (1:1000; rabbit polyclonal; Cell Signaling Technology, Danvers, MA, USA), anti-NCAM (1:10,000, rabbit polyclonal; Santa Cruz Biotech), anti-PLP1 (1:1000, rabbit polyclonal; Abcam), anti-SYNPO (1:500, rabbit polyclonal; Abcam), anti-VIM (1:1000, rabbit polyclonal; Genscript, Piscataway, NJ, USA). Twenty to fifty micrograms of proteins were used depending on the sensitivity of the specific antibody. Immunoreactivity was detected by using an HRP chemiluminescent substrate reagent kit (Invitrogen, Carlsbad, CA). A pooled sample was used to normalize the inter-gel variation between repeated runs for the same protein. Immunoreactivities of antibodies were visualized with Luminata™ Forte or Crescendo Western HRP substrate (Merck Millipore, Germany) and quantified with the Alliance 4.7 image analyser (UVItec, UK).