Co-localisation and co-immunoprecipitation of nuclear Pcolce with expPABPN1 in myotube cultures. A. Pcolce sub-cellular localisation in myotube cultures. (i) Parental IM2, and those stably expressing Ala17- or Ala10- FLAG-tagged PABPN1were co-stained for Pcolce and PABPN1. PABPN1 was detected with 3 F5-VHH llama antibody and anti-mouse Alexa 594. Rabbit-anti-Pcolce antibody was detected with Alexa 488. Nuclei are counterstained with DAPI. Scale bars equal 20 μm. Arrows indicate unfused myoblast cells. (ii) Single confocal microscope section of myonuclei in Ala17 or Ala10 cell clones. Co-localisation of the two proteins is shown in the merged image. Plots of fluorescence intensity distribution (measured across the line in the merged image) show co-localisation of Pcolce and PABPN1 in fluorescent foci (arrows). Representative myonuclei with PABPN1 foci are shown. Scale bars equal 5 μm. B. Immunoprecipitation of PABPN1 in protein extracts from parental IM2, Ala17- and Ala10- PABPN1-FLAG myotube cultures was performed with rabbit-anti-FLAG antibodies. Immunoblotting was conducted with mouse-anti FLAG antibody or rabbit anti-Pcolce. IgG HC is used as a loading control.