Anti actin mab1501

Protein samples were extracted from sponge implants (5-μm-thick cryostat sections) using RIPA buffer (Sigma, Saint Louis, MO) containing proteinase inhibitor cocktail (Roche Diagnostics, Mannheim, Germany). Samples were separated using 4–15% Mini-PROTEAN TGX precast gels (BioRad, Hercules, California). For Western blotting, the following antibodies were used: anti-MMP-2 (sc-13595, Santa Cruz Biotechnology, Dallas, Texas), anti-MMP-9 (AB19016, Millipore, Darmstadt, Germany), anti-MMP12 (AF3467, R&D systems, Minneapolis, MN), anti-actin (MAB1501, Millipore, Darmstadt, Germany).

All reagents were purchased from Sigma-Aldrich/Merk (St. Louis, MO, USA), except where specified otherwise. Anti-PARP (9542) and -caspase-3 (9662) were purchased from Cell Signaling (Beverly, MA, USA). Anti-actin (MAB1501) and anti-PGC-1α (ST1202) antibodies were purchased from Millipore (Billerica, MA, USA). The siGENOME SMART pool siRNAs and DharmaFECT transfection reagents were purchased from Thermo Scientific (Waltham, MA, USA). Dulbecco’s modified Eagle medium (DMEM)/F12 and fetal bovine serum (FBS) were purchased from GE Healthcare (Logan, UT, USA). PRO-PREP™ protein extraction solution was purchased from iNtRON Biotechnology (Seoul, Korea). Apart from pDsRed-Mito (expressed as a red fluorescent mitochondrial marker) and pEGFP-N1 (expressed as an enhanced green fluorescent protein), which were purchased from Clontech Laboratories (Mountain View, CA, USA), and pTagRFP-C (expressed as a red fluorescent protein), which was obtained from Evrogen (Moscow, Russia), all plasmids were purchased from Addgene (Cambridge, MA, USA). The luciferase reporter assay kit was purchased from BD Biosciences Clontech (Palo Alto, CA, USA).

Tissue samples were homogenized in a buffer containing 150 mM NaCl, 50 mM Tris-HCl (pH 7.5), 1 mM EGTA, 1% NP40, 0.25% sodium deoxycholate, 1 mM sodium vanadate, 1 mM NaF, 10 mM sodium β-glycerophosphate, 100 nM okadaic acid, 0.2 mM phenylmethylsulfonyl fluoride, and a 1:1,000 dilution protease inhibitor cocktail (Sigma). Protein concentration was determined using bicinchoninic acid assay and equivalent amounts of protein were resolved by LDS-(lithium dodecyl sulfate)-PAGE (4–12% gel; NuPAGE, Invitrogen). Proteins were transferred to a nitrocellulose membrane (0.2 μm, Bio-Rad) and blocked for 1 h in 5% non-fat dry milk (Bio-Rad) resolved in Tris-buffered saline, containing 1% Tween-20. Membranes were incubated overnight at 4 °C on a rocking platform with respective primary antibodies. The following primary antibodies were used: anti-Fas (05–351; clone 7C10), anti-actin (MAB1501) (Millipore, Billerica, MA, USA), anti-caspase 8 (ALX-804-448; 3B10) (Enzo Life Sciences, Farmingdale, NY, USA), anti-cleaved caspase 3 (9661), anti-PARP (9542S), anti-cytochrome c (4272), anti-HSP90 (4877), anti-Akt (9272) and anti-phospho-Akt (Thr308; 9275S) (Cell Signaling, Danvers, MA, USA), anti-cleaved-Bid (ab10640) (Abcam, Cambridge, UK), anti-Grp78 (sc-1051) (Santa Cruz Biotechnology, Dallas, TX, USA), anti-SREBP1 (557036) (BD Biosciences, Allschwil, Switzerland), and anti-BID^{53 (link)}.