Mouse antibody to β actin

Total proteins were extracted with RIPA buffer in presence of protease inhibitors (Roche). Membrane proteins were extracted using Membrane and Cytosol Protein Extraction Kit (Beyotime) according to manufacturer’s instructions. Samples were separated with SDS-PAGE and blotted on a NC membrane (Pall Corporation). Membranes were incubated using these specific primary antibodies: rabbit antibody to RAB18 (1:2000, Sigma), mouse antibody to β-actin (1:5000, Santa Cruz Biotechnology), mouse antibody to FLAG (1:1000, Sigma), mouse antibody to GAPDH (1:4000, Proteintech), rabbit antibody to RAB3GAP2 (1:2000, Novus Biologicals),mouse antibody to N-cadherin (1:1000, BD Transduction Labs), rabbit antibody to NaK ATPase (1:10000, Abcam), rabbit antibody to GRASP65 (1:5000, Abcam), mouse antibody to KDEL (1:1000, Abcam), rabbit antibody to GluR1 (1:2000, Abcam) and mouse antibody to βIII-tubulin (Tuj1) (1:5000, Sigma). We used secondary antibodies to rabbit and mouse HRP. Proteins were revealed using a chemiluminescence kit (Thermo Fisher Scientific).

Total protein was extracted using radioimmune precipitation buffer protein lysis buffer according to protocols (Beyotime, Shanghai, China). The protein content of the samples was measured by means of the BCA protein assay kit (Beyotime, Shanghai). The protein samples were denatured and separated by 10 % sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes. The nitrocellulose membranes were blocked for 1 h at room temperature in 5 % skimmed dried milk. Then the membranes were incubated with primary antibodies including: rabbit antibody to GFRα3 (1:1000, Abcam Systems, USA), rabbit antibody to artemin (1:200, Abbiotec, San Diego) and mouse antibody to β-actin (1:1000, Santa Cruz Biotechnology, USA) in TBST containing 3 % fat-free dry milk for 1 h at room temperature and overnight at 4 °C. After washing 3 times with TBST, the membranes were incubated with the secondary donkey anti-rabbit or anti-mouse IgG antibodies (1:5000, Santa Cruz, USA) at room temperature for 1 h. Finally, the immunoblots were detected using an ECL kit (Santa Cruz, USA) and visualized after exposure to X-ray films. The relative optical density ratio was calculated with the Image J software by comparison to GAPDH or β-actin.