Fig 7

(A-C) Overexpression of TMEM33 degrades MAVS in a dose-dependent manner. EPC cells were seeded in 6-well plates overnight and co-transfected with 1 ?g TMEM33-HA and 1 ?g empty vector, MAVS-Myc, MITA-Myc, or TBK1-Myc (A); TMEM33-HA (1 or 2 ?g) and 1 ?g MAVS-Myc (B) or MITA-Myc (C) for 24 h. The cell lysates were subjected to IB with anti-Myc, anti-HA, and anti-?-actin Abs. (D and E) TMEM33-?TM1 and TMEM33-?TM2 have no effect on the exogenous MAVS and MITA. EPC cells were seeded in 6-well plates overnight and transfected with the indicated plasmids (1 ?g each) for 24 h. The cell lysates were subjected to IB with the indicated Abs. (F) TMEM33 has no influences on the poly I:C/SVCV-induced transcriptions of MAVS. EPC cells were transfected with 2 ?g TMEM33-Myc or empty vector for 24 h, and then transfected with poly I:C or infected with SVCV (MOI = 1) for 24 h. Total RNAs were extracted to examine the mRNA level of mavs by qPCR. The relative transcriptional levels were normalized to the transcription of ?-actin and represented as fold induction relative to the transcriptional level in the control cells, which was set to 1. Data were expressed as mean ± SEM, n = 3. (G) Effects of inhibitors on TMEM33-mediated destabilization of MAVS. EPC cells were seeded in 6-well plates overnight and co-transfected the indicted plasmids. At 18 h post-transfection, the cells were treated with DMSO, MG132 (20 ?M), 3-MA (2 mM), or NH₄Cl (20 mM) for 6 h. The cell lysates were subjected to IB with the indicated Abs. (H) TMEM33-induced MAVS degradation is rescued by MG132 in a dose-dependent manner. EPC cells were seeded in 6-well plates overnight and co-transfected the indicted plasmids. At 18 h post-transfection, the cells were treated with DMSO or MG132 (10, 20, or 40 ?M) for 6 h. Then, the cells were harvested for IB with the indicated Abs. (I) TMEM33 promotes the ubiquitination of MAVS. EPC cells were transfected with 5 ?g MAVS-Myc, 5 ?g TMEM33-HA or empty vector, and 1 ?g Ub-HA. At 18 h post-transfection, the cells were treated with DMSO or MG132 for 6 h. Cell lysates were IP with anti-Myc affinity gel. Then the immunoprecipitates and WCLs were analyzed by IB with the indicated Abs. (J) TMEM33 mediates K48-linked ubiquitination of MAVS in vivo. EPC cells were transfected with 5 ?g MAVS-Myc, 5 ?g TMEM33-HA or empty vector, and 1 ?g Ub-HA, Ub-K48O-HA or Ub-K63O-HA. At 18 h post-transfection, the cells were treated with MG132 for 6 h. At 24 h post-transfection, cell lysates were IP with anti-Myc-affinity gel. Then the immunoprecipitates and WCLs were analyzed by IB with the indicated Abs. All experiments were repeated for at least three times with similar results.