Phospho ampk

Western blot was performed to detect protein expression as previously described (Yuan et al. 2012 (link)). Briefly, total proteins from primary rat cortical neurons were isolated using the protein buffer (Beyotime, Shanghai, China). Proteins were then separated by 10% SDS-PAGE and transferred onto PVDF membranes. After being blocked with 5% fat-free milk, the membranes were incubated at 4 °C overnight with primary antibodies against p62 (1:1,000, Abcam, Cambridge, MA, USA), Beclin-1 (1:1,000, Abcam), microtubule-associated protein 1 light chain 3 (LC3-I and LC3-II, both from Anti-LC3B antibodies, 1:1,000, Sigma-Aldrich), CaMKKβ (1:1,000, Abcam), AMPK (1:1,000, Cell Signaling Technology Inc., Danvers, MA, USA), phospho (p)-AMPK (1:1,000, Cell Signaling Technology Inc.), mTOR (1:1,000, Cell Signaling Technology Inc.), and p-mTOR (1:1,000, Cell Signaling Technology Inc.). Subsequently, membranes were incubated for 2 h at room temperature with horseradish peroxidase (HRP)-secondary antibodies in TBST. The protein was visualized using the ECL detection system (Beyotime, Shanghai, China). The band intensity was quantified with Image-Pro Plus 6.0 software.

Frozen rat esophagus tissue was homogenized in Tissue Protein Extraction Reagent (ThermoFisher, Waltham, MA, USA) using TissueLyser II (Qiagen) at 30 Hz for 5 min. Extracted protein was then quantified using the DC Protein Assay (Bio-Rad). Western blot analysis was performed as previously described [123 (link)]. Images were captured using the Bio-Rad ChemiDoc Imaging System and quantified by means of chemiluminescent immunodetection using Bio-Rad Image Lab Software version 6.1.0 with expression levels normalized to the loading control GAPDH. Immunoblotting was performed using commercially available antibodies from Abcam (Cambridge, MA, USA) and Cell Signaling Technology (Danvers, MA, USA): ABST (ab203205, 1:500), ATF-4 (CST #11815, 1:1000), CD44 (ab189524, 1:500), GAPDH (CST #2118, 1:25,000), HSP60 (CST #12165, 1:5000), IRE1α (CST #3294, 1:1000), and phospho-AMPK (CST #2535, 1:1000). Patient EAC samples with matched normal and BE tissues were collected at the University Hospital at the University of Michigan. Informed consent was obtained from patients prior to sample collection. Protein extraction and quantification were similarly performed as described above.

Human ORS and DP cells or skin tissue were lysed (RIPA lysis buffer; #20‐188; Merck Millipore). The obtained protein was then separated by 8% SDS–PAGE and transferred to a polyvinylidene fluoride membrane (Amersham). The protein transferred membranes were incubated overnight with the specific antibodies. The following antibodies were used: β‐actin (#MA5‐15739, 1:5,000; Thermo Fisher), phospho‐ERK1/2 (#9101, 1:1,000; Cell Signaling), total ERK1/2 (#9102, 1:1,000; Cell Signaling), phospho‐AMPK (#2535, 1:1,000; Cell Signaling), and total AMPK (#2532, 1:1,000; Cell Signaling). Then, the membranes were washed and incubated with anti‐rabbit IgG or anti‐mouse IgG antibodies (horseradish peroxidase‐conjugated, GTX213110, GTX213111, 1:10,000; GeneTex) at 25°C for 1 h. Antibody‐antigen complexes detected by enhanced chemiluminescent substrate (Thermo Fisher) were captured and quantified by Amersham imager 680 systems (GE Healthcare).

EZ-cytox cell viability assay kit was purchased from DoGen (Seoul, Korea). Muse cell cycle kit, Muse annexin V, and dead cell assay kit were purchased from the Millipore (Bedford, MA, USA). Gemcitabine and crystal violet solution were purchased from Sigma-Aldrich (St. Louis, MO, USA). In situ cell death detection kit, fluorescein was purchased from Roche (Mannheim, Germany). Enhanced chemiluminescence regent was purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Trizol reagent and cDNA synthesis kit were purchased from Invitrogen Inc. (Carlsbad, CA, USA). Power SYBR^® Green PCR master mix was purchased from Applied Biosystems (Foster City, CA, USA). Antibodies against CDK4, cyclin D1, p21, p27, GAPDH, PARP, caspase-3, Bcl-2, phospho-AMPK, and AMPK were purchased from Cell Signaling (Danvers, MA, USA). Phospho-JNK and JNK antibodies were purchased from Santa Cruz.

Protein concentrations were determined according to the Bradford method using BSA as standard. Cell lysates (10–20 μg per sample) were then separated by 5–20% SDS-PAGE with known molecular weight markers (Bio-Rad, California, USA) and transferred onto polyvinylidene fluoride membranes by standard procedures. Membranes were incubated in blocking solution (5% skim milk or BSA in TBS-T) for 3 h at room temperature, and hybridized overnight at 4°C with primary antibodies against vinculin (Sigma) (1:4000), AdipoR1 (Immuno-Biological Laboratories Inc., Minneapolis MN) (2 μg /ml), and also Akt (1:1000), phospho-Akt (s473 or t308) (1:1000), AMPK (1:1000), phospho-AMPK (1:1000) all from Cell Signaling Technology (CST, Danvers, MA, USA). The membranes were washed three times with TBS-T and then incubated with either anti-rabbit (Sigma) (1:10000) or anti-mouse (Abcam, Cambridge, U.K.) (1:10000) secondary antibodies for 1 h at room temperature. After washing, immunodetection was performed using an enhanced chemiluminescence solution (Clarity Max Western ECL Substrates, Bio-Rad) and scanned on Fujifilm LAS-3000 system and quantified using Image-J software (National Institutes of Health).

Anti-FLAG M2 (Cat# F1804), tubulin (Cat# T5168) and actin (Cat# A2066) antibodies were obtained from Sigma-Aldrich. The rabbit anti-ATGL antibody was obtained from Cayman Chemicals. Anti-p53 (DO-1) was purchased from Santa Cruz Biotechnology. Anti-Bcl-XL (Cat# 2762S), Anti-AMPK (Cat# 2532) and phospho-AMPK (Cat# 2535S) were obtained from Cell Signaling. The rabbit polyclonal antibody against G0S2 has been described previously [44]. Whole-cell extracts were obtained by harvesting cells and boiling in 1X Laemmli buffer. Western blotting was performed using standard protocols for SDS-PAGE and wet transfer onto nitrocellulose membranes (Bio-Rad).

d-glucose, 3-isobutyl-1-methylxanthine, dexamethasone, insulin, ethanol (EtOH), Oil-Red O, propylene glycol, dimethyl sulfoxide (DMSO), pioglitazone, metformin, dorsomorphin (an AMPK inhibitor, AI), and all other chemicals were of analytical grade and obtained from Sigma-Aldrich Inc. (St. Louis, MO, USA). Normal glucose (100 mg/dL) Dulbecco’s Modified Eagle Medium (DMEM; Catalog No.: 12320), penicillin/streptomycin (P/S), fetal bovine serum (FBS), horse serum, and trypsin-EDTA were bought from Invitrogen (Carlsbad, CA, USA). Rosiglitazone used in this experiment did not contain any inactive ingredients, and the pure compound was kindly provided by GlaxoSmithKline, Ltd. (Taipei, Taiwan). Phospho-AMPK, AMPK, phospho-ACC, ACC, phospho-AKT, AKT, PPARγ, C/EBPα, C/EBPβ, and GAPDH antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA).