Figure 5.
Association of RHAU with telomerase holoenzyme subunits. (A) TERC G4 motif dependent association of RHAU with dyskerin. Proteins from whole HEK293T cell lysates of either mock-transfected (−) cells or transiently expressing WT or G4 motif mutant (G4-MT, ΔG4) forms of TERC were immunoprecipitated with either control mouse IgGs (mIgG) or anti-dyskerin antibodies. Immunopurified RNP complexes were separated by SDS–PAGE and probed with anti-RHAU or anti-dyskerin antibodies. (B) TERC G4 motif-dependent association of RHAU with TERT. Protein immunoprecipitation experiments with anti-FLAG antibodies were performed with whole cells lysates of HEK293T cells transiently expressing TERT–FLAG protein together with WT or G4 motif mutant (G4-MT, ΔG4) forms of TERC. Immunopurified RNP complexes were separated by SDS–PAGE and probed with anti-RHAU or anti-FLAG antibodies. The asterisk denotes immunoprecipitated TERT–FLAG protein that cross-reacted unspecifically with the horsearadish peroxidase-conjugated secondary antibody. (C) Western blot analysis of comparable protein expression in input HEK293T cell lysates. (D) Protein immunoprecipitation experiments with anti-dyskerin antibodies were performed with whole cells lysates of HEK293T cells transiently expressing TERT–FLAG protein together with WT or G4 motif mutant (G4-MT, ΔG4) forms of TERC. Immunopurified RNP complexes were separated by SDS–PAGE and probed with anti-FLAG or anti-dyskerin antibodies.