Extended Data Fig. 4: In vitro effects of STK3/4 inhibition or knockdown and overexpression on BNIP3 Ser88 phosphorylation.
From: STK3/STK4 signalling in adipocytes regulates mitophagy and energy expenditure
a, Experimental scheme to analyze Ser88 phosphorylation after inhibitor treatment. b, Extracted ion chromatogram of 16O and 18O-labeled NSTLSphEEDYIER derived from control and XMU-MP-1 treated cell lysates (4 and 24 h). The precursor ion was analyzed in PRM mode, and showed the highest EIC of y5 ion among the produced product ions. c, HR-MS/MS spectra of 16O and 18O-labeled NSTLSphEEDYIER derived from control and XMU-MP-1 treated (24 h) cell lysates. d, The effect of decreased BNIP3 Ser88 phosphorylation by XMU-MP-1 treatment for 4 and 24 h. e, Experimental scheme to analyze Ser 88 phosphorylation in BNIP3-overexpressed cell after Stk4 siRNA knockdown (KD) or STK4 overexpression (OE). f, Extracted ion chromatogram of NSTLSphEEDYIER and its AQUA peptide. The precursor ion was analyzed in PRM mode, and showed the highest EIC of y8 ion among the produced product ions. g, Quantification of BNIP3 S88 phosphorylation in STK4 KD and STK4 OE. h, Myc-tagged BNIP3 was overexpressed in C3H10T1/2 cells and the cells were treated with XMU-MP-1 (3 μM) for indicated time. Myc-tagged BNIP3 was purified by immunochromatography and phospho-serine levels were detected by immunoblot with pan phospho-serine antibody (n = 3). Statistical analyses were assessed with one-way ANOVA with Tukey’s post hoc test for multiple comparisons. Each point represents a biological replicate. Data are presented as the mean ± s.e.m.
