Extended Data Fig. 3: NADPH depletion inhibits the degradation of MARCHF6 substrates.

a, CHX-chase experiments of SM in HeLa cells treated with control and two different NADK shRNAs. b–d, CHX-chase experiments of SM_ha, PLIN2_ha, and MARCHF6_3f in HeLa cells treated with control or G6PD siRNAs for 72 h. e, Same as in (b) but with endogenous SM and siRNA treatment for 48 h. In (a–e), right: quantification data with mean ± SD (n = 3 biologically independent experiments); two-way ANOVA. Significant P values are indicated in the figures. The levels of SM_ha, PLIN2_ha, MARCHF6_ha, and SM in G6PD siRNA or NADK#1 shRNA-treated HeLa cells at the beginning of chases were taken as 100%. f, Ub-affinity pull-down assay of SM_ha from the extracts of SM_ha-expressing HeLa cells. Cells were treated with control or NADK siRNA in the presence of the proteasome inhibitor MG132 (10 µM) for 12 h before being subjected to Ub-affinity pull-down assays. g, Same as in (f) but with PLIN2_ha from the extracts of PLIN2_ha-expressing HeLa cells. h, Chemical crosslinking and coimmunoprecipitation of MARCHF6-KO HeLa cells expressing \(\small {{{\mathrm{MARCHF}}}}6_{3{{{\mathrm{f}}}}}^{{{{\mathrm{C}}}}9{{{\mathrm{A}}}}}\) with anti-flag, followed by SDS-PAGE and immunoblotting with anti-SM, anti-NADK, and anti-flag. Cells were treated with control or NADK siRNAs for 72 h before being subjected to immunoprecipitation. i, Same as in (h) but with immunoblotting with anti-PLIN2. In (f–i), images are representative of at least three independent experiments.

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