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. 2013 Sep;142(3):241–255. doi: 10.1085/jgp.201311018

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© 2013 De Lange et al.

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Figure 6. — Effect of the E258K mutation on the interactions of N-terminal cMyBP-C with myosin S2 and cardiac actin. (A–H) Y2H interaction tests between the WT and E258K C1-C2 region of human cMyBP-C and the 126 distal amino acids of human myosin S2. (A) pGBKT7-53 × pGADT7-T (positive control of a robust interaction). (B) pGBKT7-S2 × pGADT7-S2 (positive control of a relatively weak interaction). (C) pGBKT7 × pGADT7 (negative control). (D) pGBKT7 × pGADT7-S2 (negative control). (E) pGBKT7-C1C2^WT × pGADT7 (negative control). (F) pGBKT7-C1C2^E258K × pGADT7 (negative control). (G) pGBKT7-C1C2^WT × pGADT7-S2 (interaction of interest). (H) pGBKT7-C1C2^E258K × pGADT7-S2 (interaction of interest). (I–N) Y2H interaction tests between the WT and E258K C1-C2 region of human cMyBP-C and α cardiac actin (ACTC1), as well as their appropriate control interaction tests. (I) pGBKT7-53 × pGADT7-T (positive control of a robust interaction). (J) pGBKT7-S2 × pGADT7-S2 (positive control of a relatively weak interaction). (K) pGBKT7 × pGADT7-ACTC1 (negative control). (L) pGBKT7-53 × pGADT7-ACTC1 (negative control). (M) pGBKT7-C1C2^WT × pGADT7-ACTC1 (interaction of interest). (N) pGBKT7-C1C2^E258K × pGADT7-ACTC1 (interaction of interest). Yeast were grown on minimal selection media lacking leucine, tryptophan, histidine, and adenine. (O) Quantitative β-galactosidase assay performed in diploid yeast containing pGBKT7-C1C2^WT × pGADT7-S2 (blue bar) and pGBKT7-C1C2^E258K × pGADT7-S2 (red bar). n = 3. Error bars indicate SEM.