Anti ampk 2532

The following antibodies used for Western blotting are listed: anti-GAPDH (MAB374, Merck Millipore, Billerica, MA, USA), anti-alpha SMA (ab5694, Abcam, Cambridge, UK), anti-vimentin (5741, Cell Signaling, Beverly, MA, USA), anti-phosphorylated AMPK (2535, Cell Signaling), anti-AMPK (2532, Cell Signaling), anti-phosphorylated mTOR (5536, Cell Signaling), anti-mTOR (ab32048, Abcam) and anti-collagen I (sc-166865, Santa Cruz, CA, USA). The chemicals used were PDGF-bb (520-BB, R&D System, Minneapolis, MN, USA), PDGF-bb(520-BB, R&D System) and n-butylidenephthalide (ALFA, Derbyshire, UK). For the in vitro experiments, 10% (w/v) n-BP/DMSO was prepared as the stock.

Cells were lysed in RIPA buffer and subjected to Western blot analysis as described previously [51 (link)]. Anti-Cox2 (ab198286), anti-Cox4 (ab16056), anti-SDHB (ab14714), anti-MFN2 (ab56889), anti-P4HB (ab137110), anti-PGC-1α (ab191838), anti-NRF1 (ab175932), and anti-TFAM (ab272885) antibodies were purchased from Abcam (Cambridge, UK). Anti-PDH (2784), anti-Acetyl-CoA Carboxylase (ACC) (3676), anti-phospho-Acetyl-CoA Carboxylase (pACC; Ser79) (3661), anti-AMPK (2532), and anti-phospho-AMPK (pAMPK; Thr172) (2531) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). An anti-actin (SC-47778) antibody was purchased from Santa Cruz (Dallas, TX, USA) Band intensities were quantified using densitometry and ImageJ software (Image J 1.52k) (NIH, Bethesda, MD, USA).

Acacetin and acacetin prodrug used in the present study were synthesized as described previously (Li et al., 2008 (link); Liu et al., 2016a (link)). Specific siRNA duplexes: control siRNA (sc-37007), Sirt1 siRNA (sc-40986), Sirt3 siRNA (sc-61555), and anti-β-actin antibody (sc-47778) were from Santa Cruz Biotechnology (Dallas, TX, United States). The primary antibodies anti-pAMPK (#2535) and anti-AMPK (#2532) antibodies were from Cell Signaling (Danvers, MA, United States); anti-Sirt1 (ab32441), anti-Sirt3 (ab217319), anti-Bcl-2 (ab182858), anti-Bax (ab32503), anti-PGC-1α (ab54481), anti-SOD1 (ab16831), anti-SOD2 (ab16956), anti-CD31 (ab9498) antibodies were from Abcam (Cambridge, MA, United States). Fluorescently–conjugated secondary antibodies (Alexa Fluor® 488, #A-11001; Alexa Fluor® 568, #A-11004; Alexa Fluor® 633, #A-21052) and Lipofectamine RNAiMAX Reagent were from Thermo Fisher Scientific (Waltham, MA, United States). A list of antibodies is shown in Supplementary Table S1.