Anti p ampk thr172

The relative hepatic mRNA levels of G6Pase, PEPCK, apo C-III, sterol regulatory element binding protein 2 (SREBP2), diacylglycerol acyltransferase-2 (DGAT2), SREBP1c, and β-actin were evaluated by the 2700 GeneAmp™ PCR System (Applied Biosystems (ABI), Waltham, MA, USA), and the primers are depicted in Table 1 (29 (link), 34 (link), 35 (link)). The livers and skeletal muscles were immediately removed and quickly homogenized with RIPA buffer (Sigma, St. Louis, MO, USA) prior to Western blotting (29 (link), 34 (link), 35 (link)). The expression levels of target genes were assessed by Western blotting as demonstrated in previous reports (27 (link), 32 (link), 33 (link)) using the following antibodies: anti-phospho-Akt (Ser⁴⁷³) (no. 9271), anti-total-Akt (no. 9272), anti-p-AMPK (Thr¹⁷²) (no. 2535), and anti-total-AMPK (no. 5831) from Cell Signaling Technology (Beverly, USA); anti-FoXO1 (forkhead transcription factor forkhead box O1) (no. ab39670), anti-PPARα (no. ab61182), anti-PPARγ (no. ab209350), anti-FAS (fatty acid synthase) (no. ab128856), and anti-β-actin (no. ab8227) from Abcam (Cambridge, CB2 0AX, UK); and goat anti-mouse IgG coupled to HRP secondary antibody (Jackson Lab., Inc., West Grove, USA) (29 (link), 34 (link), 35 (link)). Finally, these results were detected using chemiluminescence kits (Amersham Biosciences ECL^TM, Buckinghamshire, UK).

Proteins were obtained from the hearts or cell extracts and were analysed using Western blot analysis, as previously described [33 (link)]. The following primary antibodies were used: anti-LC3 (1:500; Abcam, Cambridge, MA, UK), anti-p62 (1:500; Abcam), anti-Beclin1 (1:500; Abcam), anti-AMPK (1:1000; Cell Signaling, Danvers, MA, USA), anti-p-AMPK-Thr172 (1:1000; Cell Signaling), anti-mTOR (1:1000; Cell Signaling), anti-p-mTOR-Ser2448 (1:1000; Cell Signaling), ULK1 (1:1000; Cell Signaling), and p-ULK1-Ser757 (1:1000; Cell Signaling). Targeted bands were normalized to GAPDH to confirm equal protein loading. The Western blot bands were quantified using ImageJ 3.0 software.

EMPA was purchased from El-Hikma Pharmaceutical Co., Giza, Egypt. Rotenone was purchased from Sigma Aldrich, USA. For Western blot analysis, anti-β-actin (Catalogue No. #ab8224), anti-AMPK (Catalogue No. #ab110036), anti-SIRT-1 (Catalogue No. #ab110304), and anti-β-catenin (Catalogue No. #ab32572) antibodies were purchased from Abcam, USA. Anti-pAMPK (Thr172) (Catalogue No. #2531) anti-PGC-1α (Catalogue No. #2178) antibodies were purchased from Cell Signaling Technology, USA. Anti-tyrosine hydroxylase antibody (Catalogue No. #NB300-109) was purchased from Novus Biological, USA. Anti-alpha-synuclein antibody (Catalog No.# sc-12767) was purchased from Santa Cruz Biotechnology, Inc.

RL71 (>97% purity, HPLC) was synthesized as described previously.^{14 (link)} anti-LC3B, anti-ubiqutin, anti-CHOP, anti-PARP, anti-p-JNK, anti-JNK, anti-p-PERK (Thr980), anti-p-AMPK (Thr172), anti-p-mTOR (Ser2448, 2971), anti-p-p70S6K (Thr389), anti-Cox4 and anti-cleaved caspase-3 antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). Anti-Grp78, anti-ATF4, anti-ATF6, anti-β-actin and anti-GFP antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-SQSTM1/p62, anti-ATG5 and anti-ATG7 antibodies were from Abcam (Cambridge, UK) and anti-LC3 was from Novus Biologicals (Littleton, CO, USA). GFP-LC3 plasmid was purchased from Yingrun Biotechnologies Inc. (Changsha, China). Compound C (CC) and BAPTA were obtained from Calbiochem (San Diego, CA, USA). zVAD was from Selleck Chemicals (Houston, TX, USA). Chloroquine (CQ), STO-609, SP600125 and PBA were purchased from Sigma-Aldrich (St. Louis, MO, USA). The ER-specific dye ER tracker Red, the mitochondrial specific dye MitoTraker Red CMXRos (M7512), JC-1 (T-3168), Fura-2/AM and Lipofectamine 3000 transfection reagent were purchased from Life Technologies (Grand Island, NY, USA). The Ca²⁺-ATPase activity assay kit was purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing). All of the other chemicals were purchased from Sigma-Aldrich.