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. 2019 Feb 26;10:344. doi: 10.3389/fmicb.2019.00344

Figure 6.

Figure 6

TRIM14 induces the destabilization of NP and inhibits the translocation of NP to nucleus. (A) HEK293T cells were co-transfected with RNP reconstitution system and TRIM14. Cells were treated by 100 μM CHX at 24 h after transfection and collected at the time points shown for immunoblot analysis. (B–D) Immunoblot analysis of extracts of HEK293T cells co-transfected with RNP reconstitution system and TRIM14. At 24 h post transfection, cells were treated with 8 μM MG132 (B), 5 mg/ml 3-MA (C) or 20 μM CQ (D) for 8 h, 8 h, 20 h. (E) Immunoblot analysis of extracts of HEK293T cells co-transfected with plasmids of Flag-NP, HA-Ubiquitin, Myc-vector or Myc-TRIM14 followed by immunoprecipitation with anti-Flag beads. (F,G) Immunoblot analysis of extracts of HEK293T cells co-transfected with plasmids of Flag-NP, Myc-TRIM14, HA-K48-Ubiquitin (F) or HA-K63-Ubiquitin (G) followed by immunoprecipitation with anti-Flag beads. (H) HeLa cells were transfected with HA-TRIM14 or vector then infected with WSN at an MOI of 1 at 24 h after transfection. Cells were fixed for confocal microscopy at 3 h, 4 h, and 8 h post infection. The red fluorescence showed the staining of NP and green fluorescence showed the staining of TRIM14. (I) HeLa cells were transfected with HA-TRIM14 or vector then infected with WSN (MOI of 1) at 24 h after transfection. Cells were harvested for the indicated time points after infection for immunoblot analysis.