Anti acc

Frozen adipose and liver tissues were homogenized and lysed in radioimmunoprecipitation assay (RIPA) lysis buffer containing protease and phosphatase inhibitors (Complete TM Mini and PhosSTOP TM, Roche Diagnostics, Indianapolis, IN, USA). Protein samples were separated using 10% sodium dodecyl sulfate-polyacrylamide (SDS) gels and transferred to a nitrocellulose membrane (Bio-Rad, Hercules, CA, USA). After blocking, the membranes were then incubated at 4 °C overnight with the following primary antibodies: anti-UCP-1, anti-PRDM16, anti-FGF21, anti-Sirt1, anti-PGC1α, anti-pAKT, anti-AKT, anti-pACC, anti-ACC, anti-pIRS1 Ser³⁰⁷, anti-NOV/CCN3, anti-TWIST2, anti-HIF1α, anti-β-actin (Cell Signaling Technology, Danvers, MA, USA), anti-pIR tyr⁹⁷² (Millipore, Bedford, MA, USA), anti-HO-1 (Enzo Life Sciences, Farmingdale, NY, USA). Membrane incubations were carried out using a secondary infrared fluorescent dye conjugated antibody absorbing at both 800 nm and 700 nm. The blots were visualized using an Odyssey Infrared Imaging Scanner (Li-Cor Science, Lincoln, NE, USA)) and quantified by densitometric analysis after normalization with β-actin. Results were expressed as optical density (O.D.) as previously described [19 (link)].

The antibodies used in the study were as follows: anti-ZKSCAN3 and anti-Atg4B from Abcam. Anti-human α-synuclein from Invitrogen. Anti-α-synuclein from BD. Anti-LC3B, anti-SQSTM1/p62, anti-phospho-4E-BP1 S65, anti-4E-BP1, anti-phospho-ACC S79, anti-ACC, anti-phospho-AMPKα T172, and anti-AMPKα, anti-phospho-p70S6K (Thr389), anti-p70S6K, anti-phospho-mTOR (Ser2448), anti-mTOR, anti-phospho-AKT (Thr308) and anti-TFEB from Cell Signaling Technology. Anti-phospho-JNK (Thr183/Tyr185), anti-JNK, anti-phospho-ERK1/2 (Tyr204), anti-ERK1/2, anti-phospho-p38 (Tyr182), anti-p38, and anti-AKT from Santa Cruz Biotechnology. The other antibodies used in the study were anti-β-actin (Millipore), and anti-α-tubulin (Pierce Biotechnology). SP600125 and rapamycin was purchased from Cell Signaling Technology; 3-methyladenine (3-MA), cycloheximide (CHX), anisomycin and chloroquine (CQ) from Sigma-Aldrich. Bafilomycin A1 was purchased from LC Laboratories.

The protein expression in the liver was determined through Western blotting following a previously described protocol [20 (link)]. The membranes were then incubated with primary antibody (PPARγ, IL-6, IL-1β, SIRT1, LXRα, pAMPKα, AMPKα, PPARα, pACC, ACC, FAS, SREBP1c, CPT1B, and β-actin) at room temperature for 2 h. In this study, the primary antibodies were anti-PPARγ (Cat# 2435S, 1:1000, Cell Signaling, Danvers, MA, USA), anti-IL-6 (Cat# 21865-1-AP, 1:1000, Proteintech, Rosemont, IL, USA), anti-IL-1β (Cat# 16806-1-AP, 1:1000, Proteintech), anti-SIRT1 (Cat# 9475S, 1:1000, Cell Signaling), anti-LXRα (Cat# ab176323, 1:1000, Abcam, Cambridge, UK), anti-pAMPKα (Cat# AF3423, 1:1000, Affinity, San Francisco, CA, USA), anti-AMPKα (Cat# AF6423, 1:1000, Affinity), anti-PPARα (Cat# sc-398394, 1:500, Santa Cruz, Santa Cruz, CA, USA), anti-pACC (Cat# D7D11,1:2000, Cell Signaling), anti-ACC (Cat# C83B10, 1:2000, Cell Signaling), anti-FAS (Cat# C20G5, 1:2000, Cell Signaling), anti-SREBP1c (Cat# ab28481, 1:2000, Abcam), anti-CPT1B(Cat# DF3904, 1:500, Affinity), and anti-β-actin (Cat# GTX109639, 1:10000, Genentech, San Francisco, CA, USA). The relative expression of proteins was quantified densitometrically using ImageJ software (Wayne Rasband, Madison, WI, USA), and β-actin was used as the internal control.

The paraffin-embedded tissue sections were deparaffinized with xylene and dehydrated in gradually decreasing concentrations of ethanol. For immunohistochemical analysis, the dehydrated tissue sections were immersed in 10 mM sodium citrate buffer (pH 6.0) for 5 min at 95 °C. The last step was repeated using 10 mM fresh sodium citrate solution. The sections were allowed to cool in the same solution for 20 min and then rinsed with PBS. Next, the sections were incubated with a primary antibody for 1 h at 37 °C. The primary antibodies were anti-PPAR-γ (Cell Signaling Technology, Danvers, MA, USA), anti-SREBP-1 (Abcam, Cambridge, Cambridgeshire, UK), anti-FAS (Cell Signaling Technology, Danver, MA, USA), and anti-ACC (Cell Signaling Technology). After three rounds of serial washing with PBS, the sections were processed with an indirect immunoperoxidase technique using a commercial EnVision System kit (DAKO, Carpinteria, CA, USA). The slides were examined with a Pannoramic^® MIDI slide scanner, and integrated optical density was analyzed using the i-Solution DT software (IMT i-Solution).

Cells were lysed in ice-cold RIPA buffer (50 Mm Tris-HCl (pH 7.4); 150 Mm NaCl; 1 mM EDTA; 1% NP-40; 0.25% sodium deoxycholate) with protease inhibitors (Protease inhibitor Cocktail Tablets, Roche, Shanghai, China). Proteins were separated by SDS-PAGE (Bio-Rad) and transferred to Immobilon-P membranes (Millipore, Billerica, MA, USA) and immunoblotted using the following antibodies: anti-AMPKα (Cell signaling technology, Danvers, MA, USA, #5831), anti-ACC (Cell signaling technology, #3676), anti-phospho-AMPKα (Thr172) (Cell signaling technology, #2535), phospho-ACC (Ser79) (Cell signaling technology, #11818), and anti-βactin (Sigma-Aldrich, A5441). The phosphorylation was quantified as the ratio of AMPKα and ACC to its total protein level.