Ecl substrate reagent

Western blots were performed on mouse cerebellar tissues according to standard methods [40 (link)]. Tissue lysates were prepared in RIPA lysis buffer (Millipore, Billerica, MA, U.S.A) containing protease and phosphatase inhibitors (Roche, Grenzacherstrasse, Basel, Schweiz). Protein samples were separated by gel electrophoresis, transferred to nitrocellulose membrane (Bio-Rad), and blocked with 5% albumin bovine serum (Sigma-Aldrich) or 5% milk (Bio-Rad). After incubation with primary antibodies at room temperature overnight and then secondary antibodies at room temperature for 1 h, protein signals were visualized using ECL substrate reagents (Thermo Scientific, Waltham, MA, USA). The intensities of the bands were quantified by ImageJ and normalized to the GAPDH level.

Cortex and cerebellum were homogenized separately in RIPA lysis buffer (Millipore) containing protease and phosphatase inhibitors (Roche). Tissue lysates were centrifuged at 15,000 rpm at 4°C for 20 minutes. The levels of protein in the supernatants were determined by Protein Assay solution (Bio-Rad). Protein samples were diluted with 4× loading buffer (composition: 0.02% bromophenol blue, 40% glycerol, 250 mM Tris-HCl, 5% β-mercapethanol) and RIPA lysis buffer to the required concentration and denatured at 95°C for 5 minutes. Protein samples were separated by gel electrophoresis and transferred to nitrocellulose membrane (Bio-Rad). After blocking with TBST containing 5% albumin bovine serum (Sigma) or 5% milk (Bio-Rad), membranes were incubated in p38 (rabbit, 1:1000), phospho-p38 (rabbit, 1:1000), ERK (rabbit, 1:1000), phospho-ERK (rabbit, 1:1000), JNK (rabbit, 1:1000), phospho-JNK (rabbit, 1:1000; Cell Signaling Technology), or actin (goat, 1:5000; Santa Cruz Biotechnology) antisera overnight. After rinsing in TBST, they were incubated in HRP-conjugated secondary antibodies purchased from Cell Signaling Technology or Life Technologies at room temperature for 1 h. Membranes were washed in TBST and protein signals were visualized using ECL substrate reagents (Thermo-scientific). The intensities of the bands were quantified by ImageJ and normalized to actin.

Cortical neuronal cultures were homogenized in RIPA lysis buffer (Millipore) containing protease and phosphatase inhibitors (Roche, Grenzacherstrasse, Basel, Switzerland). Tissue or cell lysates were centrifuged at 15,000 rpm at 4°C for 20 minutes. Protein levels in the supernatant were determined by Protein Assay solution (Bio-Rad, Hercules, CA, USA). Protein samples were diluted with 4x loading buffer (composition: 0.02% bromophenol blue, 40% glycerol, 250 mM Tris-HCl, 5% β-mercaptoethanol) and RIPA lysis buffer to the required concentration then denatured at 95°C for 5 minutes. Protein samples were separated by gel electrophoresis and transferred to nitrocellulose membrane (Bio-Rad). After blocking with TBST containing 5% bovine serum albumin (BSA, Sigma) or 5% milk (Bio-Rad), membranes were incubated in primary antisera overnight. After rinsing in TBST, they were incubated in HRP-conjugated secondary antibodies at room temperature for 1 hour. Membranes were washed in TBST and protein signals were visualized using ECL substrate reagents from Thermo-scientific (Waltham, MA, USA). The intensities of the bands were quantified by ImageJ and normalized with actin or GAPDH level.