L1293

Coverslips from transport and transfection assays were fixed with 3.75% formaldehyde for 15 minutes, permeabilized with 0.02% Triton-X for 5 minutes and blocked with 2% FBS and 2% BSA in PBS for 1 hour. Primary antibodies (anti-Flag M2 (mouse monoclonal, F1804, Sigma), anti-LaminA (rabbit polyclonal, L1293, Sigma), anti-KPNA7 (rabbit polyclonal, HPA031395, Sigma)) were diluted in blocking buffer and incubated at room temperature for 2 hours. Secondary antibodies (Goat Anti-Rabbit-Cy3 (115-165-144, Jackson ImmunoResearch), Donkey anti-mouse-FITC (715-225-151, Jackson ImmunoResearch)) were diluted in blocking buffer and incubated at room temperature for 1 hour. DAPI was used to stain nuclei. Images were acquired by laser scanning confocal microscopy (Zeiss 880 LSM, Carl Zeiss) at 40×, 1.3 NA oil immersion objective with Zen software (Carl Zeiss). Quantification of nuclear signal was performed with ImageJ software and statistical analysis was performed in GraphPad Prism.

The following primary antibodies at the indicated dilutions were used in this study:
Rabbit polyclonal anti‐GFP (Abcam, ab290; dilution 1:2,000); mouse monoclonal anti‐alpha‐Tubulin (Sigma‐Aldrich, T9026; dilution 1:1,000); rabbit polyclonal anti‐ASH2L (gift from Winship Herr; dilution 1:2,000); rabbit polyclonal anti‐Lamin A (Sigma‐Aldrich, L1293; dilution 1:2,000); mouse monoclonal anti‐RPB1 CTD (1PB 7C2, (Nguyen et al, 1996; dilution 1:20,000); rat monoclonal anti‐RPB1 CTD phospho Ser2 (Active Motif, 61083; dilution 1:2,000); rabbit polyclonal anti‐H3K4me3 (Abcam, ab8580; dilution 1:1,000); rabbit polyclonal anti‐H3K36me3 (Abcam, ab9050; dilution 1:1,000); rabbit polyclonal anti‐H3K9ac (Abcam, ab4441; dilution 1:1,000); rabbit monoclonal anti‐H2Bub (Cell Signaling Technology, 5546; dilution 1:5,000); rabbit polyclonal anti‐H3 (Abcam, ab1791; dilution 1:2,000), mouse monoclonal anti‐GCN5 2GC 2C11 (dilution 1:1,000; Brand et al, 2001) or 5GC 2AG (dilution 1:1,000; Krebs et al, 2010); rabbit polyclonal anti‐ZZZ3 2616 and anti‐mADA3 2678 (dilution 1:1,000; Nagy et al, 2010); rabbit polyclonal anti‐SPT20 3006 (dilution 1:1,000; Krebs et al, 2011); mouse monoclonal anti‐TRRAP 2TRR 2D5 dilution (1:1,000; Robert et al, 2006), rabbit polyclonal anti‐SGF29 2461 dilution (1:1,000) and rabbit polyclonal anti‐YEATS2 2783 (dilution 1:1,000; Nagy et al, 2010).

Laminin and haematoxylin and eosin (H & E) staining were performed as previously described [47 (link),51 (link)]. To measure the cross sectional area (CSA) of myofibres, muscle sections were stained with an anti-laminin A antibody (L1293; Sigma-Aldrich). Laminin, a cell-adhesion molecule strongly expressed in the basement membrane of skeletal muscle, was detected using an appropriate secondary antibody. Morphometric analyses were performed on sections collected from similar regions of each muscle using a Leica DMI4000 B automated inverted microscope equipped with a DCF310 digital camera. Image acquisition was controlled by the Leica LAS AF software. The ImageJ software was used to determine the CSA of 1,000 to 3,000 individual fibres from at least two different fields for each muscle section. Four to nine sections from each muscle were analysed. For histological analyses, serial muscle sections were obtained and stained in H & E following standard procedures. The number of fibres was counted and analysed using the ImageJ software.
Single myofiber isolation of hind limb muscle and nuclei immunofluorescence on single fibers was performed as previously described [8 (link)]. Nuclei of 30 individual fibres from each muscle were analysed.