Horseradish peroxidase conjugated anti mouse or anti rabbit igg

Total protein fractions from cell pellets and culture supernatants of U. maydis were prepared as described previously^{7 (link)}. Briefly, U. maydis cells were grown in CM medium to an OD₆₀₀ of 0.6. Cell cultures were concentrated and adjusted to an OD₆₀₀ of 20 with 1× sample buffer (50 mM Tris-HCl, 2% SDS, 10% glycerol, 100 mM DTT, 0.01% bromophenol blue, pH 6.8). Glass beads were then added to the samples prior to cell disruption using FastPrep-24 homogenizer (MP Biochemicals). Proteins from supernatants were TCA-precipitated and acetone-washed twice before dissolved in sample buffer. Proteins from cell pellets and supernatant fractions were separated by SDS-PAGE. Proteins were detected in Western blot analyses using mouse monoclonal anti-HA (1:10,000 dilution, Sigma Cat#H9658), anti c-Myc (1:10,000 dilution, Sigma Cat#M4439), anti-tubulin (1:2000 dilution, Merck Cat#CP06), or rabbit anti-mCherry (1:3000 dilution, BioVision Cat#5993) antibodies, and horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG (1:10,000 dilution, Cell Signaling) as secondary antibody.

Mouse heart tissue and cardiomyocytes were extracted with a Pro-Prep Protein Extraction Solution (Intron Biotechnology, Gyeonggi-Do, Korea), following the manufacturer’s instructions. Western assay was performed with specific antibodies for AdipoR1/R2 (Abcam, Cambridge, UK), phospho-Thr¹⁷² AMPK (pAMPK), total AMPK, phospho-Thr⁴⁷³ Akt (pAkt), total Akt, phospho-Ser¹¹⁷⁷ eNOS (p-eNOS), total eNOS, phospho-Ser⁴²⁸ liver kinase B1 (pLKB1), total LKB1, phospho-Ser²⁵⁶ Forkhead box protein O1 (pFoxO1), total FoxO1 (Cell Signaling Technology, Danvers, MA), arginase I/II, PPARα, TLR4, Perilipin-1/-2, 4-HNE, phosphoinositide 3-kinase (PI3K; Abcam, Cambridge, UK), PPARγ coactivator (PGC)–1α (1:2000; Novus Biologicals, Littleton, CO), CaMKKα/β, sterol regulatory element–binding protein (SREBP)–1c, phosphorylated acetyl-CoA carboxylase (pACC), total ACC, B cell leukemia/lymphoma 2 (BCL-2), BCL-2-associated X protein (BAX), GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA), iNOS (BD Biosciences, San. Jose, CA), PEX 5 (Invitrogen, Carlsbad, CA), and β-actin (Sigma-Aldrich, St. Louis, MO). After incubation with horseradish peroxidase–conjugated antimouse or anti-rabbit IgG (Cell Signaling Technology, Danvers, MA), target proteins were visualized by an enhanced chemiluminescence substrate (ECL Plus; GE Healthcare Bio-Science, Piscataway, NJ).

Total cell protein was extracted from cells using ice-cold lysis buffer (20 mM Tris pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1.0% Triton X-100, 1 tablet/10 cc buffer of PhosSTOP [Roche Applied Science, Mannheim, Germany], and protease inhibitor cocktail) and protein concentration was quantified using the Bradford method [20 (link)]. Equal amounts of protein (20 μg) were separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) and then blotting was performed as described previously [13 (link), 18 (link)]. The primary antibodies included mouse anti-human REIC/Dkk-3 (Momotaro-Gene Inc., Okayama, Japan), rabbit anti-pIRE1α (Novus Biologicals, Littleton, CO, USA), rabbit anti-human BiP, rabbit anti-human β-catenin, TBP (Cell Signaling Technology, Danvers, MA, USA), and mouse anti-human β-actin antibody (Sigma, St. Louis, MO, USA); all primary antibodies were diluted 1:2000 in Can Get Signal® (Toyobo Co., Ltd., Osaka, Japan). The secondary antibody horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG (Cell Signaling Technology) was diluted 1:5000 in 1% skim milk [7 (link)]. We quantified band densities using Image J (ver,1.53r).