Research Paper Volume 9, Issue 10 pp 2163—2189

Figure 7. SIRT4-eGFP interacts with OPA1. (A) HEK293 cells stably expressing SIRT4-eGFP, SIRT4(H161Y)-eGFP, or SIRT4(Δ28N)-eGFP were either untreated or treated with CCCP (10 µM, 2h) and thereafter subjected to OPA1 co-immunoprecipitation (IP) analysis using sepharose beads coupled anti-GFP single-domain-antibodies (nanobodies). Total cell lysates were loaded as input control (5%). CCCP treatment caused a complete proteolytic processing of L-OPA1 to S-OPA1. TACC3 was detected using specific antibodies and served as a representative negative co-immunoprecipitation control. (B) SIRT4 enzymatic activity is required for efficient interaction of SIRT4 with L-OPA1. The amount of L-OPA1 co-immunoprecipitated with SIRT4-eGFP, SIRT4(H161Y)-eGFP, or SIRT4(Δ28N)-eGFP was determined in relation to the protein input and subjected to ImageJ-based densitometric analysis. To evaluate statistical significance, two-way ANOVA followed by Tukey’s tests was performed (*p<0.05; n=4).