Figure 4. LRRC25 targets RIG‐I for autophagic degradation.

• A, B
  HEK293T cells were transfected with Flag‐RIG‐I (N), together with an empty vector or HA‐LRRC25 (75 and 200 ng). 24 h post‐transfection, cells were harvested and used to perform immunoblot analysis with the indicated antibodies (A). Total RNA in was extracted for qPCR analysis for RIG‐I (N) (B).
• C
  HEK293T cells were transfected with Flag‐RIG‐I together with an empty vector or HA‐LRRC25. After 12 h, the cells were left untreated or treated with IC poly(I:C) (5 μg/ml) for 24 h. Before harvesting, the cells were treated with MG132 (5 μM) for 4 h. Cell lysates were used for immunoblot analysis with the indicated antibodies.
• D
  HEK293T cells were transfected with an empty vector (no wedge) or increasing amounts (wedge) of deletion mutants of LRRC25, along with RIG‐I (N) and ISRE‐luc. 24 h post‐transfection, cell lysates were analyzed for ISRE‐luc activity.
• E
  HEK293T cells were transfected with Flag‐RIG‐I (N), together with an empty vector or deletion mutants of LRRC25. 24 h post‐transfection, cells were harvested and used to perform immunoblot analysis with the indicated antibodies.
• F
  HEK293T cells were transfected with plasmids for Flag‐RIG‐I (N), together with an empty plasmid or Myc‐LRRC25. 12 h post‐transfection, the cells were treated with DMSO, MG132 (5 μM), DMEM, NH₄Cl (10 mM), 3MA (2.5 mM), or CQ (20 μM) for 4 h, 4 h, 6 h, 6 h, 4 h, or 4 h, respectively. Cell lysates were used for immunoblot analysis with the indicated antibodies.
• G
  HEK293T cells were transfected with Flag‐RIG‐I together with an empty vector or HA‐LRRC25. After 12 h, the cells were left untreated or treated with IC poly(I:C) (5 μg/ml) for 24 h. Before harvesting, the cells were treated with MG132 (5 μM) for 4 h, together with DMEM, 3MA (2.5 mM), NH₄Cl (10 mM) for 6 h, 4 h, or 6 h, respectively. Cell lysates were used for immunoblot analysis with the indicated antibodies.
• H
  HEK293T cells were transfected with empty vector or HA‐LRRC25. After 12 h, the cells were left untreated or treated with IC poly(I:C) (5 μg/ml) for 24 h. Before harvesting, the cells were treated with MG132 (5 μM) for 4 h, together with DMEM, 3MA (2.5 mM), NH₄Cl (10 mM) for 6 h, 4 h, or 6 h respectively. Cell lysates were used for immunoblot analysis with the indicated antibodies.
• I, J
  Wild‐type (WT), BECN KO (I), or ATG5 KO (J) HEK293T cells were transfected with Flag‐RIG‐I (N), together with an empty vector or Myc‐LRRC25. Twenty‐four hours post‐transfection, cells lysates were harvested and analyzed by immunoblot.
• K
  HEK293T cells were transfected with control or LRRC25‐specific siRNAs. 24 h post‐transfection, the cells were co‐transfected with mCherry‐RIG‐I and eGFP‐LC3, followed with IC poly(I:C) (5 μg/ml) treatment for 24 h. The cells were then subjected to confocal microscopy analysis. Scale bar, 10 μm.

Data information: In (A, C, E–K), data are representative of three independent experiments. In (B, D), data are mean values ± SEM (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001 (Student's t‐test).