Fig. 4: Enzymatic activity of ATGL switch variants.

A Western blot analysis of ATGL switch variant expression in Expi293 cells. 5 µg protein of the whole cell lysate was separated on a 10% SDS-gel and stained with anti-Protein A Ab (top) and anti-GAPDH Ab (bottom). B, C TAG hydrolase activity detected in lysates of Expi293 cells. Activity was determined in the absence (−CGI-58, dark gray bars) or presence (+CGI-58, light gray bars) of purified full-length mouse CGI-58 from E. coli (B) or 10 µg lysate from cells expressing full-length human CGI-58 (C). EV, lysate with empty vector control, for measures of basal activity (−CGI-58, dark gray bars), the CGI-58 preparation was heat inactivated before addition. Single amino acid ATGL variants are color-coded according to the most specific binding alteration with a single partner, color code: CGI-58 green, G0S2 yellow, PLIN1 cyan, and PLIN5 blue. All samples were measured in triplicates and are shown as mean values + standard deviation. Statistical significance was determined by unpaired two-tailed t-test (*p < 0.005, **p < 0.01, ***p < 0.001). Source data are provided as a Source Data file. D Dose-dependent CGI-58 stimulation of ATGL switch variants. Data are presented as mean values, error bars indicate ± standard deviation of triplicate experiments. E TAG hydrolase activity detected in lysates of Expi293 cells as in D including the ATGL-F348E + R351L double mutant protein variant. ATGL-WT, L159A, F348E, and R351L are shown for direct comparison. Data are shown as mean values + standard deviation. Statistics as in (B and C).