Iblot 2 nc ministacks

Total RNA was isolated and purified using TRIzol reagent (Invitrogen, #15596026) according to the manufacturer's protocol. cDNA was prepared to employ a CycleScript RT premix (Bioneer, #K-2044-CFG). All primer sequences are listed in Table 1. Expression levels were calculated using a SensiFAST SYBR Lo-ROX Kit (Bioline, #BIO-94020) and a commercial detection system (BioRad, #CFX96).
Total proteins were extracted into RIPA buffer (Thermo Fisher Scientific, #89900) containing a phosphatase inhibitor (Sigma-Aldrich, #4906845001) and a protease inhibitor (Roche, #43693159001), subjected to 10–16% (w/v) Tris-glycine SDS-PAGE, transferred to PVDF membranes using Iblot 2 NC ministacks (Invitrogen, #IB23002), and the membranes blocked with 5% (w/v) skim milk. The primary antibodies were anti-pAMPK (Cell Signaling Technology, #2531S), anti-AMPK (Cell Signaling Technology, #5831S), anti-pACC (Cell Signaling Technology, #3661S), anti-ACC (Cell Signaling Technology, #3662S), and anti-GAPDH (Cell Signaling Technology, #2118S) diluted 1:1000 (primary antibodies) or 1:5000 (secondary antibodies) in TBST (Biosesang, #HT2007) containing 5% (w/v) skim milk. The membranes were then incubated with a peroxidase-conjugated anti-rabbit secondary antibody and signals quantitated using the Immobilion Western Chemiluminescent HRP Substrate (Millipore, #WBKLS0500).

Total proteins were extracted using RIPA buffer (ThermoFisher Scientific, #89900, CA, USA), containing phosphatase inhibitor (Roche, #4906845001, Penzberg, Germany) and protease inhibitor (Roche, #4693159001, Penzberg, Germany), separated by SDS-PAGE, and transferred to NC membranes using Iblot 2 NC mini stacks (Invitrogen, #IB23002, CA, USA). Following primary antibodies were used: anti-Glucose-6-phosphate isomerase (Abcam, #ab66340, MA, USA), and anti-GAPDH (Cell signaling technology, #2118, MA, USA); the antibodies were diluted 1:500~5,000 with TBST (Biosesang, #HT2007, Seongnam, Korea) containing 5% skimmed milk. The signals were detected by Immobilion western chemiluminescent HRP substrate (Millipore, #WBKLS0500, Darmstadt, Germany).

To obtain macrophage cell lysates, cells were washed once with DPBS and lysed by addition of ice-cold RIPA buffer. The suspension was incubated for 10 min on ice and subsequently transferred to a 1.5 ml Eppendorf reaction tube. After centrifugation for 10 min at 16,873g and 4°C, the lysate was transferred into a 1.5 ml fresh tube without disturbing the resulting pellet. After addition of SDS sample buffer, the lysates were incubated at 95°C for 10 min before analysis by Western blot. Protein lysates were generally separated in freshly prepared acrylamide gels (Acrylamide Kit, 20%; Bio-Rad TGX FastCast) or gradient gels (mPAGE 4–12% Bis–Tris, 10 × 8, 15-well; premade; Millipore) and blotted with the iBlot 2 system on pre-stacked membranes (iBlot 2 NC Mini Stacks; Invitrogen). Protein bands were recorded after staining with primary and secondary antibodies using different automated (cytiva, Amersham Image Quant 800 with auto mode and full dynamic range or LI-COR C-DiGit blot scanner) or classical developers.