D2z3b

Total protein was extracted from the H9c2 cardiomyocytes using ice-cold radioimmunoprecipitation assay (RIPA) buffer supplemented with phenylmethylsulfonyl fluoride (PMSF). Protein concentration was determined using a bicinchoninic acid assay (BCA) protein assay kit (Lot. No. 20190215, Solarbio, Beijing, China) according to the manufacturer’s instructions. The polyvinylidene difluoride (PVDF) membranes were incubated with the primary antibodies, including rabbit anti-MCU antibody (D2Z3B, Cell Signaling Technology, dilution: 1:1,000), rabbit anti-CBARA1/MICU1 antibody (D4P8Q, Cell Signaling Technology, dilution: 1:1,000), rabbit anti-MICU2 antibody (A12198, ABclonal, dilution: 1:1,000), and anti-beta actin monoclonal antibody (ab115777, Abcam, dilution: 1:200). The antigen–antibody bands were detected using enhanced chemiluminescence (ECL) solution and visualized using X-ray film (Beyotime Institute of Biotechnology). Quantification of bands was performed by densitometric analysis using Bio-Rad Quantity One. β-Actin served as an internal control.

HUVECs in the flow chambers were washed twice with cold phosphate‐buffered saline and lysed using RIPA buffer (ab156034; Abcam) and HaltTM protease and phosphatase inhibitor cocktail, EDTA‐free (78441; ThermoFisher Scientific). Lysates were homogenized by passing through a 25‐gauge needle and centrifuged at 14,000g for 10 min. Equal amounts of protein (20 μg/lane) were separated on 4%–12% Bis‐Tris polyacrylamide gels (WG1402BOX; ThermoFisher Scientific), transferred to a PVDF membrane (1B24001; ThermoFisher Scientific) using iBlot 2 PVDF regular stacks (IB21001; ThermoFisher Scientific), and probed with antibodies specific for MCU (D2Z3B, 1:5000; Cell Signaling), MICU1 (D4P8Q, 1:3000; Cell Signaling); MICU2 (ab101465, 1:2000; Abcam), MCUb (MBS3223833, 1:2000; MyBioSource), MCUR1 (13706, 1:3000; Cell Signaling), TOMM20 (42406S, 1:3000; Cell Signaling), and ACTB (SC47778, 1:10,000; Santa Cruz Biotechnology). Signals were visualized via chemiluminescence (SuperSignal West Pico PLUS Chemiluminescent Substrate: 34578; ThermoFisher Scientific) and quantified using ImageJ (https://imagej.nih.gov/ij/).

For KD validation, cells were seeded on 10 cm dishes, transfected with respective siRNAs and harvested 48 h post transfection. Rabbit polyclonal citrin antibody (Abcam, ab96303) at 1:1000 dilution and rabbit monoclonal MCU antibody (Cell Signaling Technology, D2Z3B, #14997) at 1:1000 dilution were used for immunoblotting. A 1:5000 dilution of goat anti-rabbit secondary antibody was used (Santa Cruz Biotechnology, sc-2054). For phosphorylated PDH assessment, cells on 10 cm dishes were incubated in experimental storage buffer for 20 min to adjust to room temperature, followed by 5 min incubation in 2 Ca²⁺ buffer. After, cells were either incubated in 2 Ca²⁺ buffer for 5 min, in 0 Ca²⁺ for 5 min, or in 0 Ca²⁺ buffer for 1 h. Following the incubation times, cells were washed with ice cold nominally Ca²⁺ free buffer and harvested on ice. Phosphorylated PDH was blotted with 1:1000 dilution of rabbit mAb P-PDH S293 (Cell Signaling Technology, E4V9L, #37115), and total PDH with 1:1000 dilution of rabbit mAb PDH (Cell Signaling Technology, C54G1, #3205). A 1:5000 dilution of goat anti-rabbit secondary antibody was used (Santa Cruz Biotechnology, sc-2054). Broad Range (10-250 kDa) Color Prestained Protein Standard ladder (NEB, P7719S) was used in all blots.