VMP1 interacts with nsp3 and nsp4, related to Figs. 4 and 5. (A) Immunostaining reveals that Myc-VMP1 colocalizes with GFP-nsp3 on the ER. Bars: 500 nm; insert, 200 nm. (B) Immunostaining reveals that Myc-VMP1 colocalizes with nsp4-GFP on the ER. Bars: 500 nm; insert, 200 nm. (C) Immunostaining reveals that Strep-TMEM41B colocalizes with GFP-nsp3 on the ER. Bars: 500 nm; insert, 200 nm. (D) Immunostaining reveals that Strep-TMEM41B colocalizes with nsp4-GFP on the ER. Bars: 500 nm; insert, 200 nm. (E) OptiPrep membrane flotation analysis of cells coexpressing GFP-nsp3 and nsp4-mCherry. PNS, post-nuclear supernatant. (F) In a GFP-Trap assay, endogenous VMP1 is immunoprecipitated by GFP-nsp3 or nsp4-GFP. (G) In a GFP-Trap assay, levels of Flag-nsp4 immunoprecipitated by GFP-VMP1 with the G195R mutation are increased, compared with WT GFP-VMP1. (H) In a GFP-Trap assay, levels of Flag-nsp3 immunoprecipitated by GFP-VMP1 or GFP-VMP1(G195R) are comparable. (I) Immunostaining reveals that GFP-VMP1(G195R) accumulates at the sites of Flag-nsp3/nsp4-mCherry puncta, as indicated by arrows. Bars: 500 nm; insert, 200 nm. (J) In a GFP-Trap assay, levels of nsp4-mCherry immunoprecipitated by GFP-nsp3 are comparable in control and Myc-VMP1-overexpressing cells. (K and L) The size of enlarged hypodermal LDs (arrows) in epg-3 mutant worms is dramatically reduced by pssy-1(RNAi) (K). Quantitative data are shown as mean ± SD (N2, n = 505; epg-3, n = 878; L). ****, P < 0.0001. Bars: 10 μm. (M) Quantitative PCR results show that the transcription of PTDSS1 or PTDSS2 is efficiently suppressed in siPTDSS1 or siPTDSS2 cells, respectively. mRNA levels of PTDSS1 and PTDSS2 are normalized by ACTB levels. (N) Immunoblotting results demonstrate that levels of p62 and LC3-I/II remain unchanged after single or double knock down of PTDSS1 and PTDSS2 in control and VMP1 KO HeLa cells. Source data are available for this figure: SourceData FS3.