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Fig. 3. HCR expression and localization in C2C7 cells. (A) Total lysates from C2C7 myogenic cells incubated in differentiation medium (DM) for the indicated times or maintained in growth medium (GM) were analysed by western blot using the indicated antibodies. ${alpha}$ -Tubulin was used to normalize the amount of protein extracts loaded in each lane. (B, top) Indirect immunofluorescence of HCR performed using purified anti-HCR rabbit polyclonal antibody in cycling (GM) and differentiated (DM) myogenic C2C7 cells. (B, bottom) Hoechst-stained nuclei of the same fields as in B, top. (C) Colocalization of overexpressed EGFP-RPB3 and Flag-HCR proteins. (left) Fluorescence of EGFP-RPB3 fusion protein; (middle) indirect immunofluorescence using anti-Flag monoclonal antibody; (right) merge of EGFP-RPB3 and Flag-HCR fluorescence signals. (D) Colocalization of endogenous RPB3 and HCR proteins. (Green) Indirect immunofluorescence using rabbit polyclonal antibody against RPB3; (red) indirect immunofluorescence using mouse polyclonal antibody against HCR; (yellow) merge of RPB3 and HCR indirect immunofluorescence signals; (blue) Hoechst-stained nuclei relative to the same field.

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