Anti laminin α1 l9393

Rat MuRF1 and murine UBE2E1 (E2E1) cDNAs were amplified by RT–PCR from either rat soleus muscles or murine C2C12 myotubes mRNA using Superscript II and Platinum Pfx DNA polymerase (Invitrogen, Carlsbad, CA, USA). cDNAs were cloned in the pcDNA3.1 expression vector (Invitrogen) for expression in mammalian cells. MuRF1 and E2E1 shRNA were produced using pLKO.1 clones purchased from Sigma (Supplementary Table S1). Oligos used for miR production (Supplementary Table S2) directed against E2E1 and the pcDNA™6.2-GW/EmGFP-miR vector were obtained from ThermoFisher Scientific. Combinations of at least 2 RNAi vectors were used for knocking down MuRF1 and E2E1.
Immunoblotting and immunohistochemistry (IHC) were performed using anti-flag (F1804), α-actin (HHF-35), PCNA (P8825), MHCI (M8421), and anti-laminin-α1 (L9393) from Sigma, St. Louis, MO, USA, E2E1 (TA309924), from Origene, Rockville, MD, USA, MHCI (clone BAF8), MHCIIa (clone N2), and MHCIIb (clone BFF3) from DSHB (University of Iowa, Iowa City, IA, USA) and anti-caspase 3 (9662) and cleaved caspase 3 (Asp175, 9661) from Cell Signaling Technology, Danvers, MA, USA.

The antibodies used for Western blot were monoclonal Anti-Proliferating Cell Nuclear Antigen (PCNA) (clone PC10 Sigma-Aldrich P8825; 1:3000), monoclonal anti-alpha-actin (Sigma-Aldrich HHF35; 1:5000), monoclonal anti-myosin heavy chain type IIa (DSHB SC-71; 1:1000), monoclonal anti-Flag (Sigma-Aldrich F1804; 1:6000) and polyclonal anti-UBE2L3 (Sigma; 1:1000). The secondary antibodies used were anti-mouse (IRDye^® 800 CW, Licor 92632210 or IRDye^® 680, Licor 92632220;), anti-goat (IRDye^® 800 CW, Licor 92632214) and anti-rabbit (IRDye^® 800 CW, Licor 92632221). The antibody used for skeletal muscle cross-sectional analysis was anti-laminin-α1 (L9393) from Sigma.
Heavy Meromyosin (HMM; Cat. # MH01) (including the first 800 amino acids of MHC plus the two pairs of light chains) and alpha-actin (Catalogue No. AKL99) purified from rabbit skeletal muscle were purchased from Cytoskeleton, Inc., Denver, CO, USA. Alpha-actin was reconstituted in General Actin Buffer (5 mM Tris-HCl pH 8.0, 0.2 mM CaCl2 and 0.2 mM ATP) supplemented with 0.2 mM ATP and 0.5 mM DTT, following the supplier’s instructions. Then 1/10th volume of Polymerization Buffer (500 mM KCl, 20 mM MgCl2, 10 mM ATP) was added to induce the polymerization of a-actin. UBE2G2, UBE2N, UBE2V2, UBE2Z and UBE2K were purchased from LifeSensors. UBE2D2 and UBE2C were purchased from Enzo Life Sciences.