GluA1/GluA2 Western Blot Analysis

Western blot analysis was performed as previously described (Inoue et al., 2014 (link)). Protein extracts (3 μg) were subjected to SDS-PAGE and separated proteins were transferred onto polyvinylidene difluoride membranes. After blocking with a solution containing 5% skim milk in PBS, the membranes were incubated with rabbit anti-GluA1 (1:2000, catalog #GluR1C-Rb-Af692, Frontier Institute, Hokkaido, Japan) or anti-GluA2 (1:2000, catalog #GluR2C-Rb-Af1050, Frontier Institute) polyclonal antibodies overnight at 4°C, then with an appropriate HRP-conjugated secondary antibody for 1 h at room temperature. Protein bands were detected using an ECL chemiluminescence detection system and an Image Quant LAS 4000 Mini (GE Healthcare, Uppsala, Sweden). For reprobing, the membranes were incubated with stripping buffer (62.5 mM Tris-HCl, 2% SDS, and 100 mM 2-mercaptoethanol) for 30 min at 50°C. The membranes were incubated with rabbit anti-transferrin receptor antibody (1:1000, catalog #ab84036, Abcam, Bristol, UK) and then processed as described above. The signal of the protein bands was quantified using ImageJ 1.46r software.