Extended Data Fig. 3: KSHV vBcl-2 regulates NM23-H2 activities.
a, IB (top) and densitometric quantification (bottom) of NM23-H2 stability in cells transduced with Vec or HA-vBcl-2 (WT/E14A) and treated with CHX (n = 3). b, Coupled pyruvate kinase-lactate dehydrogenase assay (Methods) showing vBcl-2-driven NDPK activity of recombinant NM23-H2 preincubated with GST-vBcl-2 (WT/E14A) or Vec (n = 3 independent experiments). c, in vitro assay showing that increasing amount of recombinant GST-vBcl-2 (WT or E14A) modulate histidine phosphorylation of purified His-NM23-H2 in the presence of ATP, detected by IB with anti-1-pHis antibody. Coomassie blue staining shows input of each purified proteins as indicated. d, Densitometric quantification of 1-pHis levels in (c) normalized to NM23-H2 input (n = 3 independent experiments). e-g, Representative confocal images of in HeLa cells (e) co-transfected with HA-vBcl-2 and Flag-NM23-H2 showing the distributions of vBcl-2 (green) and NM23-H2 (red) relative to organelle markers (magenta), including PDI (ER), TGN46 (trans-Golgi), TOM20 (mitochondria), and WGA (wheat germ agglutinin; plasma membrane). Scale bars, 10 μm. The percentage of vBcl-2 overlapping with each marker (f) and vBcl-2 colocalization with NM23-H2 (g) were quantified (n = 50 cells pooled from three independent experiments). Data in a,c,e are from one experiment that is representative of three independent experiments. See Source Data for uncropped data of a,c. Data in a,b,d represent mean ± s.d. analyzed by one-way ANOVA followed by Tukey’s post hoc test. Box-plot data in f,g are presented as median (center line), interquartile range (IQR; box, 25th to 75th percentiles), and whiskers extending from minimum to maximum values; and analyzed using Kruskal-Wallis with post hoc Dunn’s test. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; ns, not significant. Exact P values are provided in Source Data Extended Fig. 3.
