SingPLOS One | DOI:10.1371/journal.pone.0155645 May 13,3 /D-trp(8)-MSH Prevents LPS Effects on Skeletal MuscleTable 1. Primers for real-time PCR. Gene 18S HPRT COX-2 TNF- IL-1 CRH IGF-I IGFBP-3 LC3b Bnip-3 Gabarap1 atrogin-1 MuRF-1 MHC I MHC IIa Forward Primer (5′ to 3′) GGTGCATGGCCGTTCTTA CTCATGGACTGATTATGGACAGGAC ACCAACGCTGCCACAACT TGAACTTCGGGGTGATCG GCTGTGGCAGCTACCTATGTCTTG CGCAGCCGTTGAATTTCTTG GCTATGGCTCCAGCATTCG GGAAAGACGACGTGCATTG CAGGTTGCCTAGCAGAGGTC CAGAGCGGGGAGGAGAAC TATCCCTCCCACCAGTGCTA GAACAGCAAAACCAAAACTCAGTA TGTCTGGAGGTCGTTTCCG CCTGCAGCTCCAAGTTCAGT CCATATATTTTATCAAATCACATCCAA Reverse Primer (5′ to 3′) TCGTTCGTTATCGGAATTAACC GCAGGTCAGCAAAGAACTTATAGCC GGTTGGAACAGCAAGGATTT GGGCTTGTCACTCGAGTTTT AGGTCGTCATCATCCCACGAG GCGGGACTTCTGTTGAGG TCCGGAAGCAACACTCATCC GCGTATTTGAGCTCCACGTT TGTCCTATACACCTGACCTGTTTC GAAGCTGGAACGCTGCTC AAATAGTCTTCCTCATGGTTGTCC GCTCCTTAGTACTCCCTTTGTGAA ATGCCGGTCCATGATCACTT ATCAGCTGGTCGCATCTTTC GGTGATCAGCAGCATTTCG Solution bp 60 122 118 122 120 112 62 78 67 80 63 74 58 69doi:ten.CCL22/MDC Protein medchemexpress 1371/journal.pone.0155645.tcycle threshold 2(CT) process, with 18S and HPRT as reference genes. PCR merchandise had been separated employing agarose gel electrophoresis to confirm the solution presence and size.Western blotGastrocnemius and myotubes were homogenized in RIPA buffer (10l/ mg) with protease inhibitor cocktail, sodium deoxycolate 12.5 mM, phenylmethane sulfonyl fluoride 100 mM, sodium orthovanadate 12.5 mM and with phosphatase inhibitors (Sigma-Aldrich, Madrid, Spain). The homogenate was later centrifuged at 13000 rpm at 4 for 30 min to get rid of tissue debris. Protein concentration was determined using the Bradford protein assay with bovine serum albumin as standard. The protein extract was boiled for 5 min using a 1:1 volume of Laemmli loading buffer. Proteins (100 g from gastrocnemius or 20 g from myotubes) have been resolved by electrophoresis on 14 polyacrylamide gels under decreasing conditions, and after that transferred onto nitrocellulose membranes that had been blocked by incubation in five non-fat dry milk, 0.1 Tween (Sigma-Aldrich, Madrid, Spain), in Tris-buffered saline. Ponceau-S staining was performed to ensure equal transfer prior to blocking. Membranes were probed overnight at four sequentially with antibodies against pAktSer(473) (1:1000, Cell Signaling Technologies, antibody ID: AB_2315049), Akt (1:2000, Santa Cruz Biotechnology, antibody ID: AB_671714), p-mammalian target of rapamycin (pmTOR) (1:750, Cell Signaling Technologies, antibody ID: AB_330970), mTOR (1:1000, Cell Signaling Technologies, antibody ID: AB_10695460), microtubule-associated protein-1 light chain three (LC3A/B (D3U4C) XP1 Rabbit mAb) (1:1000, Cell Signaling Technology, antibody ID: 12741), pNF-Bp65Ser(536) (1:1000, Cell Signaling Technology, antibody ID: AB_331284, clon 7F1), pNF-Bp65Ser(276) (1:1000, Santa Cruz Biotechnology, antibody ID: AB_1128534), NF-Bp65 (C20) (1:1000, Santa Cruz Biotechnology, antibody ID: AB_632037, clon C-20), pFoXO1Ser(276) (1:750, Cell Signaling Biotechnology, antibody ID: AB_10827635), FoXO1 (1:1000, Santa Cruz Biotechnology, antibody ID: AB_640607), pFoxO3a (1:500, Santa Cruz Biotechnology, antibody ID: AB_653226), FoxO3a (D19A7) (1:750, Cell Signaling Technology, supplier Catalog no: #12829), atrogin-1 (1:1000, Santa Cruz Biotechnology, antibody ID: AB_2104267, clon H-300), MuRF1 (1:1000, Santa Cruz Biotechnology, antibody ID: AB_2287871, clon H-145) and -tubulin (1:5000, Sigma-PLOS A single | DOI:ten.CFHR3 Protein Biological Activity 1371/journal.PMID:24268253 pone.0155645 Might 13,four /D-trp(eight)-MSH Prevents.
