D6750

pAAV-EF1a-DIO-hChR2(H134R)-EYFP-WPRE was generously provided by Karl Deisseroth (Stanford Univ.). For preparation of AAV, HEK293T cells were grown in DMEM media with antibiotics and FBS. The day before transfection, four plates beyond 90% confluence from 10-cm dishes were plated onto five 15-cm dishes and incubated for 18–22 h or until 60 to 70% confluence. HEK293T cells were transfected with pAAV-DIO-ChR2-EYFP, pAAV-DJ and pHelper using jetPEI transfection reagent (QBiogene). The DNA/DMEM/PEI cocktail was vortexed and incubated at room temperature for 20 min. After incubation, the transfection mixture was added to each 15 cm dish. Transfected cells were harvested 48 h after transfection and incubated with 0.5% sodium deoxycholate (Sigma; D6750) and 50 units/ml of benzonase nuclease (Sigma; E1014) at 37°C for 1 h. After removing cellular debris by centrifuging at 3000 × g for 15 min, the supernatant was filtered through a 0.45 mm PVDF filter (Millipore). Purification of AAV- DJ particles was performed using HiTrap heparin affinity columns (GE Healthcare). For concentration of AAV, Amicon ultra-15 centrifugal filter units with a 100,000 molecular weight cutoff were used. Concentrated virus aliquoted and frozen for storage at −80°C. The final viral concentrations was 3~6 × 10¹² virus particles per ml for each AAV.

A diffusion method of chemical decellularization, adapted from previously published methods (30 (link), 31 (link)), was employed. Embryonic hearts were placed in 2 mL low retention tubes (Fisher Scientific 02-681-321), submerged with detergent and/or buffer, and placed on a rotator at 15 rpm (Thermo Fisher) for the following durations. Hearts were washed with ddH₂O for 8 h, sulfobetaine-10 (SB-10, Sigma Aldrich D4266) for 4 h, 100 mM Na/50 mM phosphate-buffered saline (PBS) for 15 min, 5% sodium deoxycholate (SD, Sigma Aldrich D6750)/sulfobetaine-16 (SB-16, Sigma Aldrich H6883) for 4 h, 100 mM Na/50 mM PBS for three 15-min periods, SB-10 for 1.75 h, 100 mM Na/50 mM PBS for 15 min, 5% SD/SB-16 for 3 h, 50 mM Na/10 mM PBS for two 15-min periods, deoxyribonuclease I (DNase I, Sigma-Aldrich D4527) solution for 12 h (with no rotation), and 50 mM Na/10 mM PBS for 15 min. Decellularized hearts, in fresh 50 mM Na/10 mM PBS solution, were stored at −80°C until further use.

HEK293T-HSPB8-V5 were transfected with untagged pCi-HSPB8 constructs using Lipofectamine3000/P3000 reagent and following the manufacturers’ instruction (Invitrogen, L3000001). Forty-eight h after transfection, HEK293T-HSPB8-V5 cells were harvested and then centrifuged at 100 x g for 5 min at 4°C. Pellets were resuspended in RIPA (0.15 M NaCl [Sigma-Aldrich, S3014], 0.8% sodium deoxycholate [Sigma-Aldrich, D6750], 100 mM sodium orthovanadate [Sigma-Aldrich, S6508], 50 mM NaF [Sigma-Aldrich, S7920], 5 mM sodium iodoacetate [Sigma-Aldrich, I2512], 0.05 M Tris HCl, pH 7.7, 10 mM EDTA [Sigma-Aldrich, E6758], pH 8, 0.08% SDS [Sigma-Aldrich, L3771] and Triton X-100 [Sigma-Aldrich, X100]) buffer with protease inhibitors cocktail (Sigma-Aldrich, P8340) and then centrifuged at maximum speed. For the immunoprecipitation, SureBeads Protein A Magnetic Beads (Bio-Rad, 161–4013) were used, following the manufacturers’ instructions. An anti-BAG3 (Abcam, ab47124) and an anti-V5 (D3H8Q) (Cell Signaling Technology, 13202) antibodies were used for co-immunoprecipitation. Samples were then run on SDS-PAGE and western blot was performed (see western blot and FRA section).

Following passage, cell pellets were washed with PBS. Cell pellets for mass spectrometry (MS) analysis were lysed in 8 M urea dissolved in 100 mM ammonium bicarbonate in sterile distilled water plus 2% sodium deoxycholate. Cell pellets for Western blot (WB) analyses were lysed on ice for 15–20 min with an equivalent volume of RIPA buffer (1% tergitol-type NP-40 (NP40; 9016-45-9, ICN Biomedicals, Aliso Viejo, CA, USA), 0.25% deoxycholic acid (D6750; Sigma, St. Louis, MO, USA), 1mM ethylenediaminetetraacetic acid (EDTA; #104245S, BDH), 150 mM sodium chloride (NaCl; BP358-212, Thermo Fisher Scientific, Waltham, MA, USA) and 50 mM Tris-HCl b, pH 7.4 (Tris; BP152-1, Thermo Fisher Scientific)). All samples were sonicated for 10 s and then centrifugated at 13,000 RPM (MSE, Heathfield, UK; MSB010.CX2.5 Micro Centaur) for 5 min at 4 °C to pellet any insoluble material, and their protein concentrations were determined via Pierce™ BCA Protein Assay Kit (#23227; Thermo Fisher Scientific). Absorbance was measured at 562 nm with a FLUOstar Omega microplate reader (BMG LABTECH), and a standard curve was generated from a plot of the average blank-corrected absorbance for each standard versus its concentration.