Fig. 2: Flavivirus-NS2A targets FAM134B for degradation.
From: AMFR-mediated Flavivirus NS2A ubiquitination subverts ER-phagy to augment viral pathogenicity
a, b Western blotting analysis of HEK293T cells co-transfected for 48 h with indicated Myc-FAM134B and Flag-tagged ZIKV viral protein-expressing plasmids or empty vector. The red font highlighted the ZIKV-NS2A protein. c Western blotting analysis of A549 cells transfected for 48 h with increasing amounts of Flag-NS2A. d Huh7.0-GFP-FAM134B transfected for 48 h with increasing amounts of Flag-NS2A, determined by flow cytometry. e Huh7.0-GFP-FAM134BR142A cells were infected with recombinant virus ZIKV-HA-NS2A at a MOI of 1 for 72 h and assessed confocal microscopy analysis. The plus sign (+) represents virus-infected cells marked by HA-NS2A. Scale bars, 10 μm. f Quantification of mean intensity of GFP-FAM134BR142A, related to (e). g HEK293T cells were co-transfected with Flag-NS2A and HA-tagged ER-phagic receptor-expressing plasmids or empty vector. Cells were harvested for coIP using anti-HA beads. The asterisk (*) represents the indicated ER-phagy receptors. h Huh7.0 cells were either mock-infected or infected with ZIKV-HA-NS2A at a MOI of 1 for 72 h, and then cells were harvested for coIP using anti-HA beads and analyzed by western blotting. The asterisk (*) represents the co-immunoprecipitated endogenous FAM134B, and the band of HA-NS2Aα detected by western blotting represented a truncated form of NS2A20. i HEK293T cells were co-transfected with HA-FAM134B and Flag-tagged flavivirus-NS2A-expressing plasmids or empty vector. Cells were harvested for coIP using anti-flag beads. The asterisk (*) represents the indicated flavivirus-NS2A. j Schematic diagram of FAM134B mutants. k HEK293T cells were co-transfected with FAM134BWT and mutants with Flag-NS2A or empty vector. Cells were harvested for coIP using anti-flag beads. The asterisk (*) represents the indicated FAM134BWT and mutants. l, m Huh7.0-GFP-FAM134B△NTD were transfected for 48 h with increasing amounts of Flag-NS2A (l) or infected with ZIKV at a MOI of 1 (m) for 72 h, determined by flow cytometry. Error bars of (d, f, l, m) present as the mean ± SEM (d, l, m: n = 3; f: n = 32); Statistical analysis of (d, l, m) was performed using one-way ANOVA with Tukey’s multiple comparisons test and (f) was performed using two-tailed unpaired t-test analysis; Source data are provided as a Source Data file.
