Fig. 3

The effect of PINK1-mediated phosphorylation of LETM1 on liposomes calcium release activity in vitro. a About 1 µg purified bacterial full-length His-LETM1-WT, T175E, and T192E were incorporated into liposomes and subjected to calcium release assay. Control (Con) indicates no proteins incorporated. b (top panel) Quantification of calcium release activity measured by rate of calcium release and calculated as change of fluorescence unit (∆F). n = 6. (bottom panel) The liposome samples in a were analyzed by WB with anti-LETM1 antibody to show loading of proteins. c HEK293 cells were co-transfected with pAdtrack LETM1 (AdLETM1)-WT or T192A and Adtrack control GFP (AdGFP, WT, T192A) or PINK1 (AdPINK1, pWT, pT192A), and AdLETM1-T192E only for 1 day. The FLAG-LETM1 proteins were isolated by IP with anti-FLAG, probed with pT192 or anti-LETM1 by WB. The experiment was replicated three times. d HEK293 cells were co-transfected with AdLETM1-WT or T192A and AdGFP or AdPINK1, and AdLETM1-T192E only for 1 day. The FLAG-LETM1 proteins were isolated by IP with anti-FLAG and eluted by 3XFLAG peptide. The eluted proteins were subjected to calcium release assay using artificial liposomes. Control is without protein incorporated. e (top panel) Quantification of calcium release activity of d measured by rate of calcium release and calculated as change of fluorescence unit (∆F). n = 6. (bottom panel) The liposomes samples in d were subjected to WB with anti-LETM1 antibody to show similar loading of proteins