Nonidet p 40

Cultured cardiomyocytes were lysed on ice for 30 min inlyses buffer containing (in mM) 50 Tris - HCl (pH 7.4), 1 EDTA, 100 NaCl, 20 NaF, 3 Na₃VO₄, 1 PMSF, and with 1% Nonidet P-40, and protease inhibitor cocktail (Roche, Basel, Switzerland). The lysates were centrifuged at 12000 × g for 15 minutes, and the supernatant was recovered. After denatured, equal amounts of lysate proteins were separated on 10% SDS/PAGE gels, followed by transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA, USA). After blocking, the proteins were probed with the appropriate primary antibodies against ASIC1, ASIC2a, ASIC3, TRPV1 (Alomone labs, Jerusalem, Israel.ASC-014, ASC-012, ASC-018, ACC-030, all in 1:200 dilution) and OGR1 (Santa Cruz Biotechnology, USA. sc-98437). Membrane-bound primary antibodies were detected using proper secondary antibodies conjugated with horseradish peroxidase. Immunoblots were developed on films using the enhanced chemiluminescence technique37 (link)38 (link) (SuperSignalWest Pico; Pierce Chemical Co., Rockford, IL, USA).

Co-IP assay was performed as previously described [16 (link)]. For transfection-based Co-IP assays, 2 × 10⁵ cells were transfected with the indicated plasmids using Lipofectamine 2000 (Invitrogen, CA, US), lysed in 500 μL of lysis buffer (50 mM Tris at pH 8.0, 500 mM NaCl, 0.5% Nonidet P-40, 1 mM dithiothreitol, and protease inhibitor tablets from Roche Applied Science), and immunoprecipitated with 20 μg anti-FLAG (Sigma-aldrich, CA, US) overnight at 4 °C. The beads were washed three times with the lysis buffer and eluted in SDS sample buffer. The eluted immunocomplexes were separated by SDS-PAGE, followed by western blotting with indicated antibodies according to the standard procedures. For detecting endogenous protein interactions, cells were lysed in 500 μL of lysis buffer and immunoprecipitated with indicated antibody or control serum (Santa Cruz Biotechnology, CA, US). After extensive washing with the lysis buffer, the immunoprecipitates were resolved by SDS-PAGE, followed by western blot analysis.

50 μg of total protein extract was denatured in 0.1% SDS, 0.1 M 2-Mercaptoethanol buffer, for 5 min at 95 °C. The samples were incubated for 4 h at 37 °C with N-glycosidase F (Roche Applied Science) in deglycosylation buffer (400 mM Sodium phosphate mono/dibasic, pH 7.4) with the addition of protease inhibitor (Complete; Roche Applied Science) and 0.8% v/v Nonidet P40 (Roche applied Science) as described by Viapiano Lab (Boston, MA, USA), 2007. Bovine fetuin (Sigma Aldrich, Inc., St. Louis, MO, USA) was used as a control glycoprotein. Enzyme reactions were subsequently analyzed by SDS-PAGE and western Blot analysis.

Single cells were individually transferred into 200-μl tubes using a mouth pipette. The single cells were lysed in 7 μl of soft buffer (500 mM KCl, 100 mM Tris-HCl (pH = 8.3), 1.35 mM MgCl₂, 4.5 mM DTT, 0.45% Nonidet P-40 (Roche, 11332473001), 0.18 U SUPERase-In (Applied Biosystems, AM2694), and 0.36 U RNase-inhibitor (Applied Biosystems, AM2682) for 30 min at 4 °C, and then the lysate was vortexed for 1 min at room temperature. All RNAs were released, whereas the nucleus remained intact. The lysed single cell was then centrifuged at 1 000× g for 5 min to leave the nucleus at the bottom of the tube. The 4 μl of lysis product supernatant was carefully removed and added to another 200-μl tube containing spike-in RNA (ERCC, Ambion) and reverse transcriptase. This fraction was used for transcriptome analyses, whereas the remaining 3 μl of lysis solution (containing the nucleus) was used for genome (CNVs) and methylome analyses. The upper 4 μl of lysis solution was reverse-transcribed with poly T primers, and the cDNA was amplified as previously described^{29 (link)}. Protease was added to the bottom 3 μl lysis solution, and the DNA was added with 60 fg of unmethylated lambda DNA (Fermentas). The released naked DNA was then digested and bisulfite-converted using the scRRBS method^{19 (link)}.