Anti mff

Dulbecco’s modified Eagle’s medium (DMEM), F-12 (1:1), and bovine serum (BS) were purchased from Gibco (Grand Island, NY). Fetal bovine serum (FBS) was purchased from Corning (Corning, NY). Melatonin, doxorubicin, H2DCF-DA, intracellular ATP assay kits, N-acetyl cysteine, and 4-P-PDOT (4-phenyl-2-propionamidotetralin) were purchased from Sigma-Aldrich (St. Louis, MO). MTT thiazolyl blue and D-luciferin were purchased from Duchefa Biochemie (Haarlem, the Netherlands). 4’,6-Diamidino-2-phenylindole (DAPI), enhanced chemiluminescence reagent, antibodies against GAPDH, E2F1, c-Myc, and voltage-dependent anion channel (VDAC), MitoTracker red, and control IgG antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-AMPKα2 and anti-AMPKα1 antibodies were purchased from R&D Systems (Minneapolis, MN). MitoTracker green and anti-phospho-H2A.X (S139), anti-phospho-AMPKα (Thr172), anti-phospho-acetyl-CoA carboxylase (ACC) (S79), anti-ACC, anti-phospho-mitochondria fission factor (MFF) (S146), and anti-MFF antibodies were purchased from Cell Signaling Technology (Danvers, MA). MitoSOX Red was purchased from Invitrogen (Carlsbad, CA). The FITC-annexin V apoptosis kit was purchased from DB BioScience (San Diego, CA). The Total OXPHOS Rodent WB Antibody Cocktail was purchased from Abcam (Cambridge, MA).

Western blotting was performed as described previously [20 (link)]. Whole-cell lysates were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene difluoride (PVDF) membrane. After incubation with the appropriate antibodies, bound antibodies were detected using chemiluminescent HRP substrate (Millipore, St. Louis, MO, USA). The blots were exposed and quantified using an Alliance Mini 4M (UVITEC Cambridge, Cambridge, UK). The primary antibodies were as follows: Anti-MFN1/2, anti-OPA1, anti-DRP1, anti-MFF, anti-GRP78/BiP, anti-CHOP, anti-phospho-eIF2a, anti-caspase-3 (Cell Signaling Technology, Danvers, MA, USA), and anti-β-actin (Santa Cruz Biotechnology, Dallas, TX, USA). The secondary antibodies were anti-rabbit IgG-HRP (Cell Signaling Technology) and anti-mouse-IgG-HRP (Calbiochem, Darmstadt, Germany).

All biochemical reagents were purchased from Sigma-Aldrich unless otherwise indicated. Antibodies against, JNK1 (SC-1648, 1:2500), Dicer (SC-30226, 1:2500), Akt (SC-8312, 1:2500), phospho-Akt (Ser473) (SC-7985-R, 1:2500), PGC-1α (SC-13067, 1:2500), β-actin (SC-130656, 1:5000), and β-tubulin (SC-9104, 1:5000) were from Santa Cruz Biotechnology. Anti-Acc (3662, 1:2500), anti-phospho-Acc (Ser79) (11818, 1:2500), Anti-AMPKα (5832, 1:2500), Anti-phospho-AMPKα (Thr172) (2535, 1:2500), anti-AMPKβ (4250, 1:2500), anti-phospho-AMPKβ (Ser108) (4181, 1:2500), anti-phospho-ULK1 (Ser555) (5869, 1:2500), anti-phospho-ULK1 (Ser317) (12753, 1:2500), anti-ULK1 (8054, 1:2500), anti-phospho-MFF (Ser146) (49281, 1:2500), anti-MFF (86668, 1:2500), anti-Ago2 (2897, 1:2500), anti-Ago1 (5053, 1:2500), anti-S6 Ribosomal Protein (2217, 1:2500) and anti-phospho-JNK (Thr183/Tyr185) (1:2500), anti-Albumin (4929, 1:2500), anti-Citrate Synthase (14309, 1:2500), Anti-AMPKα1 (2795, 1:2500) were purchased from Cell Signaling Technology. Anti-IRS1 (1:2500) and anti-phospho-IRS (Ser307) (07247, 1:2500) antibodies were purchased from Upstate Biotechnology. Anti-AMPKα1 (32047, 1:2500) was purchased from Abcam.