Anti ampkα1

Anti-AMPKα1 and anti-actin antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-Gli1, anti-Shh and anti-HA antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

For Western blot analysis, polypeptides in whole cell lysates were resolved by SDS-PAGE and transferred to nitrocellulose membrane filters. Proteins were detected with a 1:1000 or 1:5000 dilution of primary antibody using an enhanced chemiluminescence (ECL) system. Images were acquired using the Chemidoc-it 410 imaging system (UVP, Upland, CA, USA) and LAS4000 system (GE Healthcare, Uppsala, Sweden). The following primary antibodies were used: anti-AMPK α1 (Cell Signaling Technology, Beverly, MA, USA), anti-phospho-AMPK α1 (Cell Signaling Technology), anti-LC3 (MBL international, Watertown, MA, USA), anti-p62 (MBL international), anti-acetyl-CoA carboxylase (ACC) (Cell Signaling Technology), anti-phospho acetyl-CoA carboxylase (p-ACC) (Cell Signaling Technology), and anti-actin (ABM, Richmond, BC, Canada).

For western blot analysis, polypeptides in whole cell lysates were resolved by SDS-PAGE and transferred to PVDF membrane filters. Proteins were detected with a 1:1000 or 1:5000 dilution of primary antibody using an enhanced chemiluminescence (ECL) system. Images were acquired using the LAS4000 system (GE Healthcare, Uppsala, Sweden) and Chemidoc-it 410 imaging system (UVP, Upland, CA, USA). The following primary antibodies were used: anti-telomerase (Abcam, Cambridge, UK, ab32020), anti-AMPKα1 (Cell Signaling Technology, Beverly, MA, USA, #2532), anti-acetyl-CoA carboxylase (ACC) (Cell Signaling Technology, #3676), anti-phospho acetyl-CoA carboxylase (p-ACC) (Cell Signaling Technology, #3661), anti-c-Myc (Santa Cruz Biotechnology, Santa Cruz, CA, USA, sc788) and anti-actin (ABM, Richmond, BC, Canada, G043).

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.

For western blotting, samples in SDS sample buffer were resolved on SDS-PAGE gels and then transferred to nitrocellulose membranes prior to blocking in TBST with 5% (w/v) milk powder or 3% (w/v) bovine serum albumin and probing with primary and secondary antibodies (GE Healthcare). Primary antibodies were: anti-Myc (Sigma, #C3956), anti-FLAG (Sigma, #F3165), anti-HUWE1 (Novus Biologicals, #NB 100-652), anti-PCF11 (Bethyl Laboratories, #A303-706A), anti-GAPDH (Cell Signaling Technology, #2118), anti-TRIM28 (Abcam, #ab22553), anti-AMPKα1 (Cell Signaling Technology, #2795), and anti-MMS19 (Proteintech, #16015-1-AP) and Tb-anti-GST (Invitrogen, #PV3551). Secondary antibodies were: Donkey Anti-Rabbit IgG (GE Healthcare, NA934V) and Sheep Anti-Mouse IgG (GE Healthcare, NA931V). FLAG and TRIM28 primary antibodies were used at a dilution of 1:10,000 and Tb-anti-GST antibody was used at a dilution 1:720 and other primary antibodies were used at a dilution of 1:1000. All GE Healthcare secondary antibodies were used at a dilution of 1:5000. Protein signal was visualized after addition of ECL detection reagent (GE Healthcare) according to manufacturer’s instructions.