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. 2023 Nov 13;24(12):e57528. doi: 10.15252/embr.202357528

Figure 5. RNF144A interacts with STING.

Figure 5

  • A
    Wild‐type (WT) and Rnf144a‐deficient (KO) MEFs were left untreated or treated with IFN‐β (100 ng/ml) for 4 h before real‐time PCR assays.
  • B
    Wild‐type (WT) and Rnf144a‐deficient (KO) BMDMs (top) or MEFs (bottom) were infected with HSV‐1 (MOI = 1) for 24 h. The supernatants were then collected and subjected to ELISA assays.
  • C, D
    HEK293T cells were transfected with indicated plasmids. At 24 h after transfection, immunoprecipitation (IP) and immunoblot (IB) assays were performed as indicated.
  • E, F
    PMA‐THP1 cells were infected with HSV‐1 (MOI = 1) (E) or transfected with HT‐DNA (1 μg/ml) (F) for the indicated periods and then the cell lysates were subjected to immunoprecipitation (IP) and immunoblot (IB) assays as indicated.
  • G
    Rnf144a‐deficient (KO) MEFs were transfected with Flag‐RNF144A. At 24 h after transfection, the cells were treated with HSV‐1 (MOI = 1), HSV60 (1 μg/ml), or left untreated for 8 h, and then in situ PLA assays were performed to examine the colocalization of RNF144A and STING. The RNF144A‐STING complex is visualized in red; nuclei are shown in blue. Scale bar, 10 μm.
  • H
    HeLa cells were transfected with plasmids expressing HA‐STING and Flag‐RNF144A. At 24 h after transfection, HeLa cells were transfected with HSV60 (1 μg/ml), poly(dA:dT) (1 μg/ml), or left untreated for 8 h. Immunofluorescence assays were performed using anti‐HA (green) and anti‐Flag (red). Nuclei were stained with DAPI. Scale bar, 10 μm. The plot of pixel intensity along the green line is shown in the right panel. Pearson's correlation coefficient was calculated using ImageJ software. Rr, Pearson's correlation coefficient.
  • I
    A schematic diagram of full‐length STING (top). TM, transmembrane; CTD, carboxy‐terminal domain; CTT, carboxy‐terminal tail. HEK293T cells were transfected with the indicated plasmids. At 24 h after transfection, immunoprecipitation (IP) and immunoblot (IB) assays were performed as indicated (bottom).
  • J
    A schematic diagram of full‐length RNF144A (top). RING, really interesting new gene; IBR, in between RING; TM, transmembrane. HEK293T cells were transfected with the indicated plasmids. At 24 h after transfection, immunoprecipitation (IP) and immunoblot (IB) assays were performed as indicated (bottom).
  • K
    HEK293T cells were transfected with the indicated plasmids. At 24 h after transfection, immunoprecipitation (IP) and immunoblot (IB) assays were performed as indicated.

Data information: Two‐tailed unpaired Student's t‐test, n.s., not significant (P > 0.05). Data shown are representative of at least three independent biological replicates. In (A), each data point represents a technical replicate. In (B), each data point represents an independent biological replicate. Error bars are presented as mean ± SD.

Source data are available online for this figure.