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Fig. 7 ATG5 is vital to TMEM47-induced degradation of MAVS and STING. (A and B) The interaction between TMEM47 and ATG5. EPC cells seeded in 10-cm2 dishes were transfected with the indicated plasmids (5 µg each). After 24 h, cell lysates were IP with anti-Flag (A) or anti-Myc (B) affinity gel. Then the immunoprecipitates and WCLs were analyzed by IB with anti-Flag, anti-Myc, and anti-HA Abs, respectively. (C) ATG5 promotes the degradation of MAVS and STING by TMEM47. EPC cells were seeded in six-well plates overnight and co-transfected with the indicated plasmids. At 24 h post-transfection, the cell lysates were subjected to IB with the anti-Myc, anti-HA, anti-Flag, and anti-β-actin Abs, respectively. (D) Validation of knock-down efficiency of ATG5. EPC cells seeded in six-well plates were transfected with 1 µg ATG5-Flag, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and 1 µg shATG5#1-PLKO.1, shATG5#2-PLKO.1, or control (Con). EPC cells transfected with plasmids encoding control (Con) or shATG5 (#1 or #2). At 24 h post-transfection, the cell lysates were subjected to IB with the anti-Flag, anti-HA, anti-ATG5, and anti-β-actin Abs. (E and F) Knockdown of ATG5 rescues the degradation of MAVS and STING by TMEM47. EPC cells were seeded in six-well plates overnight and co-transfected with the indicated plasmids. At 24 h post-transfection, the cell lysates were subjected to IB with the anti-Myc, anti-ATG5, anti-HA, and anti-β-actin Abs, respectively. (G–L) Knockdown of ATG5 restores the inhibition of MAVS or STING-induced IFN activation by TMEM47. EPC cells were seeded in 24-well plates overnight and then transfected with 250 ng IFNφ1pro-Luc, 250 ng pcDNA3.1-TMEM47, 250 ng pcDNA3.1-MAVS or MITA, 250 ng shATG5#1-PLKO.1 or shATG5#2-PLKO.1, and 50 ng pRL-TK (G and H). Luciferase activities were monitored at 24 h after transfection. The promoter activity is presented as RLU normalized to Renilla luciferase activity. EPC cells seeded in six-well plates overnight were transfected with indicated plasmids. At 24 h after transfection, total RNAs were extracted to examine the mRNA levels of cellular ifn and vig1 (I–L). The relative transcriptional levels were normalized to the transcription of β-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. Asterisks indicate significant differences from the control (*P < 0.05). All experiments were repeated for at least three times with similar results.