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. Author manuscript; available in PMC: 2021 Mar 17.
Published in final edited form as: Nat Immunol. 2020 Jul 20;21(8):868–879. doi: 10.1038/s41590-020-0730-5

Figure 4. STEEP is essential for STING trafficking from ER to Golgi.

Figure 4

(a) Illustration of the in vitro membrane budding reaction. (b) Immunoblot analyses of budded material from the in vitro membrane budding reactions using cytosolic fractions from WT or STEEP-deficient THP-1 cells and microsomes from STEEP-deficient THP-1 cells. (n = 3 biologically independent experiments). (c) HEK-293T cells were transfected as indicated and probed with mouse anti-FLAG and either rabbit anti-calreticulin (ER marker) or anti-GM130 (Golgi marker). Cells were analysed by ImageStream. (n = 3) (**P = 0.0013, **P = 0.0030, up to down). (d) ER- and Golgi-enriched pellets from mock- or cGAMP-treated WT and STEEP-deficient HeLa cells were fractionated, and immunoblotted for STEEP, STING, GM130 (Golgi), and Sec61B (ER) (n = 2 biologically independent experiments). (e) WT and STEEP-deficient THP-1 cells stimulated with cGAMP for 10min were stained with anti-Sec24, anti-STING, and anti-calreticulin. For quantification of Sec24 foci, 10, 13, 11, and 18 cells from the WT-Mock, STEEP KO-Mock, WT-cGAMP, and STEEP KO-cGAMP, respectively were counted in a blinded fashion. Representative data from one experiment are shown (n = 3 biologically independent experiments). Statistical analysis used: two-tailed unpaired t test with welch’s correction. Each data point represents one cell and are shown as means +/- st.dev. (ns P = 0.15, **P < 0.000001, nsP = 0.41, **P < 0.000001, left to right). (f) HEK-293T cells transfected with STING, VSVG, and STEEP as indicated for 24 h were subjected to ImageStream analysis for colocalization of STING and VSVG with ER and Golgi. The data are shown as Golgi/ER (n = 3) (ns P = 0.18, **P = 0.00050, left to right). (g) FLAG-tagged Sar1 was transfected into WT or STEEP-deficient Hela cells for 24 h, and stimulated with cGAMP (100 nM, 30 min) Cells were probed with rabbit anti-calreticulin (ER marker) and mouse anti-FLAG. (n = 3) (**P = 0.0000040, **P = 0.000012, left to right). (h) HEK-293T cells were transfected as indicated. ER-enriched pellets precipitated with calcium chloride were immunoblotted for GFP, HA, GM130 (Golgi), and Sec61B (ER). (n = 3 biologically independent experiments). (i) GFP-tagged ALPS (GFP133) was co-transfected with the indicated vectors into HEK-293T cells for 24 h. Fixed cells were probed with rabbit anti-calveticulin (ER marker). (n = 3), and analyzed by ImageStream (**P = 0.00080). For data from ImageStream analysis (panels c, f, g, and i), each data point represents the percent of the positive cells from one representative sample and are shown as means +/- st.dev. Statistical analysis of data in panels c, f, g, and i was performed using two-tailed Student’s t-test.