Investigating PSMC5 Protein Interactions

Neuro 2A cells were transfected with wildtype or mutant forms of PSMC5. Two days after transfection, cells were washed in PBS, lysed in RIPA buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.25% sodium deoxycholate, 10 mM sodium butyrate, protease inhibitor cocktail). Lysates were split and incubated with either nonimmune IgG (Sigma) or anti-FLAG antibodies (Sigma) for 3 hr at 4°C. Immunoprecipitation was performed with Protein G beads (Invitrogen) as described [19 (link)]. Briefly, immunoprecipitated proteins were subjected to SDS-PAGE and analyzed by Western blotting using anti-FosB/ΔFosB antibody (Cell Signaling Technology) based on published protocols [7 (link)]. For in vivo protein binding assays, we used purified nuclear fractions from punch-dissected NAc of mice after chronic cocaine treatment (20 mg/kg IP daily for 7 days, with mice used 24 hr after the last injection). Co-immunoprecipitation from nuclear fractions was performed using the Nuclear Complex Co-IP kit (Active Motif) following the manufacturer’s instructions. The following antibodies were used: MYC or ß-actin, Cell Signaling Technology (Danvers, MA), PSMC5 and histone H3, Abcam (Cambridge, MA), CBP, p300 and BRG1, Santa Cruz Biotechnology (Santa Cruz, CA), and FLAG M2, Sigma.