Wg1402box

20–25 µg of protein lysates (either from liver or from epididymal white adipose tissue) were separated by SDS-PAGE (4–12% Bis-Tris acrylamide gels, WG1402BOX, Thermo Fisher), and electro-transferred to immunoblot PVDF membranes (1620177, Biorad) according to standard procedures. Membranes were blocked (5% w/v nonfat powdered milk) for 2 h followed by overnight incubation in a primary antibody mixture (murine ACBP, ab231910, Abcam). After overnight incubation, membranes were washed, followed by 1 h incubation in horseradish peroxidase (HRP)-labeled secondary antibody mix (4050-05, Southern Biotech). Membranes were washed and revealed in the ImageQuant™ LAS 4000 (GE Healthcare). β-actin primary antibody (ab49900, Abcam) was used as loading 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 western blotting, 10 μg (human central nervous system lysates) or 20 μg (zebrafish lysates) of protein was loaded on a Bis-Tris 4–12% gradient SDS-PAGE (WG1402BOX, Invitrogen, Thermo Fisher Scientific) in MOPS-SDS running buffer (J62847.K2, Alfa Aesar, Haverhill, MA, USA), electrophoresed at 150 V for 60 min, and transferred to a nitrocellulose membrane (GE10600001, Semidry transfer, Biorad, Hercules, CA, USA). Membranes were blocked with 5% non-fat dried milk (A0830.1000, AppliChem, Darmstadt, Germany) in phosphate-buffered saline (PBS) 0.1% Tween-20 (PBST). Primary antibodies and the corresponding dilutions are listed in Supplementary Table 2 (Online resource). Secondary antibodies were goat anti-rabbit IgG-HRP or goat anti-mouse IgG-HRP (1:10 000, P044801-2 and P044701-2, polyclonal, Dako). Blots were developed with SuperSignal West Pico or Dura plus ECL reagent (34580 and 34075, Thermo Fisher Scientific). Digital images were acquired using the Amersham Imager 600 (GE Healthcare, Chicago, IL, USA). All blots were stripped (21063, Restore Western Blot Stripping Buffer, Thermo Fisher Scientific) of bound antibodies and reprobed with GAPDH to control for equal protein loading. Band intensities were measured using ImageJ and were normalized to GAPDH.