Supplementary Figure 5: Ezh1α and Ezh1β isoform expression levels in adult tissues, and specific antibody characterization.
From: A cytosolic Ezh1 isoform modulates a PRC2–Ezh1 epigenetic adaptive response in postmitotic cells
a. PCR performed on C2C12 myotubes cDNA with primers specific for Ezh1β coding sequence. The amplicon is detected at the expected molecular weight (1740 bp); M: 1kb ladder, 1: Ezh1β amplicon, 2: no template. (Independent experiments: n=1). b. Expression levels of genes encoding Ezh1α, Ezh1β and Ezh2 in different mouse tissues (St: stomach, Lu: lung, B: brain, Li: liver, Spl: spleen and Sk.M: skeletal muscle). Data were normalized on Gapdh expression. Mean is indicated (mice: n=2, plotted with single values). c. Western blots detecting EGFP or Ezh1β protein in total extract of NIH3T3 transfected or not transfected with Ezh1α-YFP or Ezh1β-YFP. Ezh1β C-term and EGFP antibodies were used for detection. Actin was used as the loading control. (Independent experiments: n=2). d. Immunoprecipitation with Ezh1 β C-term antibody and western blot in NIH3T3 total extract transfected with Ezh1α-YFP or Ezh1β-YFP. Ezh1β C-term and EGFP antibodies were used for detection. (Independent experiments: n=2). e. Western blots detecting HA or Ezh1αin NIH3T3 total extract transfected or not transfected with Ezh1β-EGFP or Ezh1α-HA. Ezh1 antibody from Raphael Margueron’s lab (Ezh1α RM) and EGFP antibodies were used for detection. Actin was used as loading control. (Independent experiments: n=1). f Co-immunoprecipitation (co-IP) and western blot analyses for Ezh1β and Eed in myotube nuclear extracts. Isotype-matched IgG represent a control antibody used for IPs. Eed isoform is specified. (Independent experiments: n=2). g. Co-immunoprecipitation (co-IP) and western blot analyses for Ezh1β, Ezh1α and Suz12 in cytosolic and nuclear extracts from myotubes. Isotype-matched IgG is the control antibody used for IPs. (Independent experiments: n=2).
