Non immune igg

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.

Mice received single intraperitoneal injections of recombinant human HMGB1 (10 μg per mouse; Sigma-Aldrich)14 (link) or anti-HMGB1 neutralizing polyclonal antibody (600 μg per mouse; Shino-Test Corporation)37 (link) at 1 h before ischemia induction, concurrent with the start of electroacupuncture pretreatment. Negative control mice were injected with saline instead of rhHMGB1 or with non-immune IgG (Sigma Aldrich) instead of anti-HMGB1 antibody.

Type I collagen hydrogel aggregates were fixed at day 21 in 4% paraformaldehyde, followed by dehydration, paraffin embedding, sectioning and staining with hematoxylin/eosin (H&E), alcian blue and ALP (all Sigma) according to previously published protocols [33 (link)]. Alternate sections were used for immunohistochemistry. The following antibody treatments were used: type II collagen (COL II) (pepsin (1 mg/ml; Sigma)/monoclonal anti-COL II antibodies (Acris Antibodies GmbH, Hiddenhausen, Germany)); chondroitin-4-sulfate (CS4) (chondroitinase ABC (5 U/mL; Sigma)/monoclonal anti-CS4 antibodies (Millipore GmbH, Schwalbach, Germany)); type X collagen (COL X) (0.25% trypsin; Sigma)/polyclonal anti-COL X antibodies (Calbiochem, Bad Soden, Germany)). Visualization of the immunostainings was performed by treatment with Advance™ HRP link and Advance™ HRP enzyme (Dako, Hamburg, Germany) followed by diaminobenzidine staining (DAB kit; Sigma). Thereafter, the slides were counterstained with hemalaun (Merck, Darmstadt, Germany). Controls with non-immune IgG (Sigma) were performed for all immunohistochemical analyzes.