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. 2021 Jul 20;12:4427. doi: 10.1038/s41467-021-24724-2

Fig. 1. MARCH8 downregulates M2 from the cell surface.

Fig. 1

a Infection of HEK293T cells with Lenti-GFP reporter viruses pseudotyped with VSV-G, IAV HA and NA or IAV HA, NA and M2 that were produced in control or MARCH8-expressing HEK293T cells. GFP-positive cells were scored with flow cytometry. b Production of Lenti-GFP reporter viruses pseudotyped with IAV HA and NA or IAV HA, NA and M2 in HEK293T cells without or with MARCH8, followed by western blot. c HEK293T cells were co-transfected with M2, Flag-tagged-GST, and increasing amounts of MARCH8, followed by western blot. d HEK293T cells were transfected with M2 and 50 or 100 ng of wild-type MARCH8 (WT) or the W114A mutant MARCH8 (W114A), followed by western blot. e–h HEK293T cells transfected with either a control vector or the MARCH8 were infected with WSN virus (MOI = 2). The cells were lysed for western blotting (e). M2 expressed on the non-permeabilized cell surface was measured by flow cytometry (f), and relative MFIs of surface M2 were shown in (g). Immunostaining of M2 and MARCH8 was shown in (h). M2 localization in control cells is indicated with arrows and in MARCH8-expression cells is indicated with stars. i–k HEK293T cells transfected with control siRNA or siRNAs against MARCH8 (siMARCH8 #1 and siMARCH8 #2) were cultured for 72 h, then infected with WSN virus (MOI = 2) for 6 h. The cells were lysed for western blotting (i). M2 expressed on the non-permeabilized cell surface was measured by flow cytometry (j), and relative MFIs of surface M2 were shown in (k). In a, g, and k, the values shown are normalized mean ± SD (n = 3), **P < 0.001, n.s., not significant, unpaired two-tailed Student t-test.