Rabbit anti mfn 1

Immunoblot analysis was carried out on 30 embryos per group and harvested cells. Briefly, samples were lysed in radioimmunoprecipitation assay (RIPA) buffer with protease inhibitor and phosphatase inhibitor. Sample aliquots containing equal amounts of protein were then boiled in sample loading buffer and separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), after which they were transferred onto polyvinylidene fluoride (PVDF)membranes (Immobilon-P, Millipore). Subsequently, membranes were blocked with 3% BSA (fatty-acid free) in TBS buffer containing 0.1% Tween 20 (TBST) at 4 C overnight, rinsed in TBST and probed with the following primary antibodies at 4 C overnight: rabbit anti-Drp 1 (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-Mfn 1 (1:1000; Santa Cruz Biotechnology), rabbit anti-Mfn 2 (1:1000; Santa Cruz Biotechnology) and mouse anti-actin (1:5000; Santa Cruz Biotechnology). The blot then was washed with TBST and
incubated with the following secondary antibodies at 4 C overnight: goat anti-mouse (1:4000; Thermo Scientific, Rockford, IL, USA) and goat anti-rabbit (1:4000; Thermo Scientific). Finally, the membranes were visualized using enhanced chemiluminescence detection reagent (Advansta, Menlo Park, CA, USA) according to the manufacturer's instructions.

Human EPCs were lysed with Pro-prep (iNtRON biotechnology, Korea). Whole cell lysates (25 µg) were separated by either 8% or 15% SDS-PAGE, after which membranes were blocked with 5% skim milk for 1 h at 25℃. Membranes were probed with specific antibodies, and proteins of interest were visualized with HRP-conjugated secondary antibodies (Thermo Fisher Scientific Inc., MA, USA) and Luminata Crescendo HRP substrates (Millipore, MA, USA). Primary antibodies against mouse anti-HIF-1A (1:1000), mouse anti-DLP1 (1:1000, all above from BD Biosciences, NJ, USA), rabbit anti-phospho-DRP1 (Ser616) (1:1000), rabbit anti-phospho-DRP1 (Ser637) (1:1000, Cell Signaling Technology, Inc., MA, USA), rabbit anti-Fis1 (1:1000), rabbit anti-OPA1 (1:1000, all above Abcam, Cambridge, UK), rabbit anti-MFN1 (1:1000), and mouse anti-beta-actin (1:2500, all above Santa Cruz Biotechnology, CA, USA) were used.