Endoa2

Protein of cardiomyocytes or heart tissues was lysed in lysis buffer (Beyotime Biotechnology, Shanghai, CN). Protein concentrations were determined by BCA protein assay kit (Beyotime Biotechnology, Shanghai, CN). The SDS-PAGE was used to separate the samples and then transferred the samples onto the PVDF membrane. The membrane was blocked with TBST containing 5% non-fat dry milk at room temperature for 2 h, then washed with TBST for 3 times, followed by incubating the membrane with the specific primary antibody against cleaved caspase-3 (Cell Signaling, Boston, MA, 1:1000, Cat. No.: 9661), CHOP (Cell Signaling, Boston, MA, 1:1000, Cat. No.:2895), IP₃R (Abcam, MA, USA, 1:1000, Cat. No.: ab108517), ERO1α (Novus, USA, 1:1000, Cat. No.: NB100-2525), EndoA2 (Santa Cruz Biotechnology, Dallas, USA, 1:200, Cat. No.: sc365704), Bcl-2 (Proteintech, Chicago, USA, 1:1000, Cat. No.: 26593-1-AP), Bax (Proteintech, Chicago, USA, 1:1000, Cat. No.: 50599-2-lg), GRP78 (Proteintech, Chicago, USA, 1:1000, Cat. No.: 11587-1-AP) at 4 °C overnight. The next day, the membranes were incubated with secondary antibody (Cell Signaling, Boston, MA) conjugated to horseradish peroxidase for 1 h. Blots were developed using a Immobilon Western Chemiluminescent HRP Substrate kit (Millipore) and molecular band intensity was determined by densitometry with Image J program (NIH, Maryland, USA).

The Co-IP test was evaluated in accordance with previously provided instructions [24 (link)]. Transfected HEK293 cells or DRG tissues were lysed in cold Co-IP RIPA buffer. The lysates were centrifuged, and 5% of each supernatant was taken for the input sample. The remaining supernatants were incubated with 5–10 μg EndoA2 (mouse, Santa Cruz, USA, sc-365704), Piezo2 (rabbit, 1:200, Alomone, Israel, APC-090) or His (mouse, Santa Cruz, USA, sc-8036) antibody at 4 °C overnight and then with protein A/G beads (GE Healthcare, UK) at 4 °C for 4 h. The immunoprecipitated samples were denatured and prepared for immunoblotting. Immunoprecipitation was performed with antibodies against Piezo2 (rabbit, 1:1000, Novus, USA, NBP1-78,624; rabbit, 1:200, Alomone, Israel, APC-090), TACAN/Tmem120a (rabbit, 1:1000, Bioss, China, bs-19952R), ASIC2 (rabbit, 1:1000, Bioss, China, bs-4915R), ASIC3 (rabbit, 1:1000, Abcam, USA, ab190638), KIF5B (rabbit, 1:2000, Abcam, USA, ab167429), KIF5A (rabbit, 1:1000, Abcam, USA, ab5628), KIF17 (rabbit, 1:1000, Bioss, China, bs-3527R), KIF3A (goat, 1:100, Santa Cruz, USA, sc-18745), KIF3B (rabbit, 1:1000, Bioss, China, bs-17085R), His-tag (rabbit, 1:1000, Cell Signaling Technology, USA, 12,698) or myc-tag (rabbit, 1:2000, Bioss, China, bs-23166R). The precipitant was washed, denatured and prepared for immunoblotting.