Co-immunoprecipitation Assay

HEK293T cells were co-transfected with Flag-GluA2 and GFP, GFP-Spastin, and its phosphomimetic and dephosphomimetic mutants. The co-IP assay was performed after transfection for 48 h, as per a previously described method (Cheng et al., 2022). HEK293T cells were lysed with a cold immunoprecipitation (IP) lysis buffer (Beyotime, Shanghai, China; 25 mM Tris-Cl pH 7.4, 100 mM NaCl, 1 mM ethylenediaminetetraacetic acid, 0.5% NP40) supplemented with protease inhibitor cocktail (Roche, Basel, Switzerland) for 30 min. The lysates were harvested and centrifuged at 12,000 rpm at 4°C for 20 min. Cell extracts were determined using the bicinchoninic acid assay (BCA) and incubated with anti-GFP agarose beads (KT Health, Shenzhen, China) at 4°C for 3 h. Following this, the beads were collected and washed twice with IP lysis buffer and once with IP wash buffer with 0.05% NP-40 in PBS. The immune complexes were collected and eluted six times with IP wash buffer. The samples were analyzed by Western blotting using anti-Flag and anti-GFP antibodies.