Figure 2.

STING interacts with FADS2 in the absence of pro-inflammatory stimulation

(A) Left: experimental scheme. Right: silver-staining of immunopurified Flag- and HA-tagged Sting (F/HA-Sting) separated on SDS-PAGE. Numbers on the left: molecular weight in kDa.

(B) Simplified schematic representation of the LA (omega-6 [Ω6], yellow) and ALA (omega-3 [Ω3], blue) fatty acid (FA) desaturation pathway, leading to the generation of LC-PUFAs. Where Fads2 is indicated is where the Fads2-dependent delta-6 desaturase (Δ6D) activity is required. Where Fads1 is indicated is where the Fads1-dependent delta-5 desaturase (Δ5D) activity is required. ALA, α-linolenic acid; LA, linoleic acid; DGLA, dihomo-γ-linolenic acid; AA, arachidonic acid; EPA, eicosapentaenoic acid; DHA, docosahexaenoic acid.

(C) Flag immunoprecipitation was performed on whole-cell extracts (WCEs) from MEF^Sting−/− expressing F/HA-Sting or not. Inputs and eluates from Flag-immunoprecipitated F/HA-Sting were analyzed by western blot (WB) using indicated antibodies.

(D) Immunoprecipitation was performed using a Sting-specific antibody on WCEs from MEF and MEF^Sting−/−. Inputs and immunoprecipitated material were analyzed by WB using indicated antibodies.

(E) As in (D), except that WCEs from MEF^Sting−/− expressing F/HA-Sting or not were used.

(F) Flag immunoprecipitation was performed on WCEs from MEFs expressing F-Fads2 or not. Inputs and eluates from Flag-immunoprecipitated Flag-Fads2 (F-Fads2) were analyzed by WB using indicated antibodies.

(G) Immunofluorescence analysis of MEF^Sting−/− stably expressing F/HA-Sting using anti-Fads2 and anti-HA antibodies and DAPI nuclear staining. BF, bright field. Pearson’s correlation coefficient values for co-localization of Fads2 and Sting in MEF cells. Pearson’s correlation coefficients were calculated from four randomly selected areas.

(H) As in (C), except that WCEs from MEF^Sting−/−, MEFs expressing F/HA-Sting, or MEFs expressing Flag-tagged STING (STING-F) or HAQ-STING (HAQ-STING-F) were used.

(I) As in (C), except that WCEs from MEFs expressing F/HA-WT-Sting or F/HA-StingΔTM were used.

(J) As in (G), except that cells were stimulated (DMXAA) or not (DMSO) for 30 min prior to immunofluorescence analysis. Pearson’s correlation coefficient values for co-localization of Fads2 and Sting in MEF cells treated with DMXAA or not. Pearson’s correlation coefficients were calculated from four randomly selected areas in each group.

All graphs present means ± SEM. p values were determined by Student’s t test. ^∗∗p < 0.01.

Related to Table 1 and Figure S2.