Myogenin sc 576

Primary antibodies were sourced as follows: lamin A/C (N-18, sc-6215, Santa Cruz), SUN2 [kind gifts from Dr Didier Hodzic, University of Washington, US and (67 (link))], emerin (NCL-EMERIN, Novacastra), GFP (ab290, ab13970, Abcam), GAPDH (sc-25778, Santa Cruz), β-actin (A5316, Sigma), α-tubulin (ab52866, Abcam), phospho-p44/42 MAPK (pERK1/2) (4370, Cell Signalling Technology), p44/42 MAPK (tERK1/2) (9102, Cell Signalling Technology), V5 (R96025, Invitrogen), nesprin-1 (MANNES1A and MANNES1E (52 (link)), generated against the C-terminus of the nesprin-1 giant), myogenin (sc-576, Santa Cruz), myosin (clone A4.1025, against all isoforms expressed by MYH1, Alexis Corporation), HA (ab1424, Abcam), KLC-1/2 (63–90, a kind gift from Prof Scott Brady, University of Illinois at Chicago, USA). Alexa fluorophore (488/546/647)-conjugated secondary antibodies were from Invitrogen. HRP-conjugated secondary antibodies were from Amersham. IF staining was performed as described previously (13 (link)). In particular, to further define the subcellular localisation for GFP-tagged nesprin-1α₂, the transfected cells were fixed by 4% paraformaldehyde/PBS, then permeabilized using either 0.001% digitonin/PBS or 0.5% NP40/PBS.

Protein levels were assessed by Western blotting. Total proteins were lysed in Tris-NP40 (100 mM Tris, 0.7% NP40, pH 7.4) and measured using the Bio-Rad protein assay. 20 μg of protein were electrophoresed onto 10% SDS-PAGE gels and blotted onto nitrocellulose membrane. Membranes were probed with antibodies raised against Myogenin (sc-576, Santa Cruz Biotechnology, diluted at 1:400), all MyHC isoforms (M7523, Sigma, diluted at 1:1000) and α-Tubulin (DM1A, Cell Signaling, diluted at 1:6000). Signals were revealed using a Clarity^TM Western ECL Substrate kit (Bio-Rad), and proteins were visualized by enhanced chemiluminescence using the ChemiDoc Touch Imaging System (Bio-Rad) and quantified with Image Lab™ Touch Software (version 5.2.1).