Figure 6: BARS activates LPAATδ and this activation is required for post-Golgi carrier formation. | Nature Communications

Figure 6: BARS activates LPAATδ and this activation is required for post-Golgi carrier formation.

From: Golgi membrane fission requires the CtBP1-S/BARS-induced activation of lysophosphatidic acid acyltransferase δ

Figure 6

Quantification of phosphatidic acid (PA) production in the LPAAT assay for post-nuclear supernatants from HeLa cells transfected with: (a) empty Flag-vector (Ctr) or LPAATδ–Flag (LPAATδ) and with BARS siRNAs for 48 h, and with the last 12 h with siRNA-resistant replacement BARS–YFP-encoding vector (BARS re-expression); (b) LPAATδ–Flag (LPAATδ) and/or with BARS siRNAs and/or LPAATδ siRNAs. Post-nuclear fractions were incubated with immunopurified BARS (Purified BARS) for 30 min at 25 °C before LPAAT assay (as indicated). (c) Quantification of PA production in the LPAAT assay for post-nuclear supernatants from HeLa cells transfected with empty Flag-vector (Ctr) or LPAATδ–Flag (LPAATδ) for 48 h or with LPAATδ siRNAs for 72 h. The anti-BARS polyclonal antibody (Anti-BARS IgG) or anti-preimmune-IgG (Anti-Preim IgG, as control) was incubated with the indicated post-nuclear fraction for 30 min at 25 °C before LPAAT assay. (d) Quantification of PA production in the LPAAT assay for post-nuclear supernatants from HeLa cells transfected with empty Flag-vector (Ctr) or LPAATδ–Flag (LPAATδ) and with BARS siRNAs for 48 h. Post-nuclear fractions were incubated with HPLC-purified BAC (BAC+) or with buffer alone (Buffer+) for 30 min at 25 °C before LPAAT assay (as indicated). (e,f) Quantification of PA production in the LPAAT assay for post-nuclear supernatants from HeLa cells transfected with: (e) empty Flag-vector (Ctr) or LPAATδ–Flag (LPAATδ) for 48 h and the last 12 h with BARSS147A–YFP, BARSD355A–YFP or BARSwt–YFP (as indicated); (f) empty Flag-vector (Ctr) or LPAATδ–Flag (LPAATδ) for 48 h and the last 12 h with BARSwt–YFP, BARSD355A–YFP, BARSS147A–YFP or BARSS147D–YFP (as indicated). Cells were infected with VSV, subjected to TGN-exit assay and post-nuclear fractionations were prepared 10 min after the shift to 32 °C temperature-release block. The dashed line indicates the level of endogenous LPAAT activity not associated with LPAATδ (see text for details). Data are means±s.d. of three independent experiments. *P<0.05, **P<0.01, ***P<0.005 versus control (Student’s t-test). See also Supplementary Fig. 7 for LPAATδ and BARS (wild type and mutants) expression levels in post-nuclear samples.

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