Anti mfn2

Cardiac tissue samples were divided into non‐ischaemic (remote) and ischaemic areas and added to lysis buffer. Protein concentration was determined using a Bio‐Rad protein assay kit (Bio‐Rad Laboratories), the proteins being added to the loading buffer.19 The proteins were then transferred to nitrocellulose membranes with transfer buffer. Skimmed milk (5% solution) was used to block the membranes for 1 hour at room temperature. The nitrocellulose membranes were exposed overnight to anti‐LDLR, anti‐PCSK9, anti‐Bax, anti‐Bcl2, anti‐caspase 3, anti‐connexin43 (Cx43), anti‐connexin43 phosphorylated at Ser368, anti‐Drp1, anti‐Mfn2 and anti‐VDAC (Cell Signalling technology, Danvers, MA, USA). Subsequently, the membranes were washed and incubated with horseradish peroxidase conjugated with anti‐rabbit IgG (Cell Signaling Technology). Finally, the bands were detected and were used to determine protein expression.19

The 20 μg aliquots of cytoplasmic and mitochondrial fractions were mixed with SDS-loading buffer and boiled at 95 °C for 3–5 min. Equal amounts of proteins were loaded and electrophoretically separated using SDS-PAGE (Pulilai Co, Beijing, China). The separated proteins were transferred to nitrocellulose membranes and the membranes then incubated at 4 °C overnight with the following primary antibodies: anti-PINK1 (1:1000, ab23707, Abcam, Boston, MA, USA), anti-parkin (1:1000, ab77924, Abcam), anti-LC3B (1:1000, #3868, Cell Signaling Technology, Danvers, MA, USA), anti-P62 (1:1000, #39749, Cell Signaling Technology), anti-Tomm20 (1:1000, #42406, Cell Signaling Technology), anti-COX IV (1:1000, ab33985, Abcam), anti-Drp1 (1:1000, #5391, Cell Signaling Technology) anti-Mfn2 (1:1000, #9482, Cell Signaling Technology), anti-OPA1 (1:1000, #80471, Cell Signaling Technology), anti-cleaved caspase-3 (1:1000, #9661, Cell Signaling Technology) or anti-β-actin (1:5000, ab8226, Abcam). The membranes were then incubated with the appropriate secondary antibodies for 1 h at room temperature. The protein bands were visualized using enhanced chemiluminescence (Amersham Bioscience, Uppsala, Sweden) and quantified using Image J software (NIH).

Hank's balanced salt solution (HBSS), Horse serum (HS), Minimum Essential medium (MEM), Neurobasal medium, Fetal bovine serum (FBS), L-glutamine (L-Gln), Penicillin-streptomycin, Antibiotic-antimycotic (AA), B27 supplement, Alex Fluor 488-conjuated IgG, Alex Fluor 555-conjuated IgG, DAPI, Prolong Gold, JC-1, Alexa Fluor® 488 Annexin V/Dead Cell Apoptosis Kit, TrypLE™ Express, MitoSOX™ Red and Power SYBR® Green PCR Master Mix were purchased from Life technologies (Carlsbad, CA). Cysteine, CaCl₂, Papain, DNase I, Poly-L-lysine, Glutamate, Cytosine-1-β-D-arabinofuranoside (AraC), MTT powder and Bovine serum albumin (BSA) were purchased from Sigma (Saint Louis, MO). Anti-Tom 20 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-Drp1, anti-Mfn2 and anti-PSD95 were purchased from Cell signaling (Boston, MA). Anti-NDUFS8 was purchased from GeneTex (Irvine, CA). Wizard® Genomic DNA Purification Kit was purchased from Promega (Madison, WI). Triton X-100 was purchased from USB (Cleveland, Ohio). Anti-Tuj1 was purchased from Covance. Anti-SDHA, Anti-CORE2 and Anti-COX1 were purchased from Abcam. IRDye-conjugated secondary antibody was purchased from LI-COR Biosciences (Lincoln, NE). Rabbit polyclonal anti-pDrp1(S637) antibody was developed using phosphopeptide (Cys-Pro-Val-Ala-Arg-Lys-Leu-pSer637-Ala-Arg-Glu-Gln-Arg-Asp) of Drp1.

Anti-Tim23 (1/1,000), anti-Tom20 (for western blots, 1/1,000, for immunofluorescence, 1/200), anti-Bcl2-L-13 (1/1,000) (Proteintech), anti-cytochrome c (1/1,000) (BD biosciences), anti-Bcl2-L-13 (1/500), anti-GAPDH (1/1,000), anti-Fis1 (1/1,000), anti-α-tubulin (1/1,000) (Abcam), anti-LC3B (for western blots, 1/1,000, for immunofluorescence, 1/100), anti-HA (for western blots, 1/1,000, for immunofluorescence, 1/200), anti-Drp1Ser637 (1/500), anti-Drp1Ser616 (1/1,000), anti-Mfn2 (1/1,000), anti-cleaved caspase 3 (1/1,000) (Cell Signaling Technology), anti-ATP synthase subunit beta (for western blots, 1/1,000, for immunofluorescence, 1/200) (Life Technologies), anti-Drp1 (for western blots, 1/1,000, for immunofluorescence, 1/100), anti-Opa1 (1/1,000) (BD Transduction Laboratories), anti-FLAG (for immunofluorescence, 1/200) (Sigma), anti-Mfn1 (1/1,000) (Abnova), anti-ubiquitin (for immunofluorescence, 1/200) (Enzo Life Science), anti-Phospho-Serine/Threonine (Upstate), anti-mCherry (1/500) (Clontech) and anti-RFP23 (link) (1/1,000) antibodies were used in this study.