Figure 5

Western blot analysis of fusion proteins derived from MYBPC3 c.3331−26T>G minigenes. MYBPC3 reference (REF, Plasmid 1446) or mutant (MUT, Plasmid 1447) minigenes were transiently transfected into HeLa cells in triplicate as indicated on the top of each lane. Cell lysates derived from the same experiment and processed in parallel were analyzed by Western blot to detect the presence of FLAG-MYBPC3 and MYBPC3-Myc fusion proteins with monoclonal anti-FLAG (a) or anti-Myc (b) antibodies. Cells transfected with the REF plasmid expressed the expected normal MYBPC3 fusion protein of about 24 kDa encoded by the minigene, detected with both anti-tag (FLAG and Myc) monoclonal antibodies. The corresponding normal MYBPC3 fusion protein was not detected with either of the two antibodies in cells transfected with the MUT plasmid. A very faint band of about 16 kDa could be observed only in the MUT lanes with anti-FLAG, but not with anti-Myc, which may correspond to a poorly translated product from the misspliced cRi30 transcript. Each PVDF membrane was stained for total protein with Amido Black (AB) after immunostaining. Full size Western blot film and membrane images including the protein ladder used are presented in Supplementary Fig. S10. The anti-FLAG Western blot film displayed a strong immunological signal for the MYBPC3 fusion protein (containing three consecutive FLAG epitopes at the N-terminal protein end for increased detection sensitivity) encoded by the REF minigene, even at the lowest film exposure of about three seconds shown here. Similar results displaying weaker bands were obtained in an additional experiment using a lower amount of the anti-FLAG antibody, also presented in Supplementary Fig. S10 including different film exposures. NTR non-transfected cells.