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

Fig. 5. MARCH8 ubiquitinates and leads to M2 degradation in lysosomes.

Fig. 5

a, b Rescue of M2 expression from MARCH8-induced degradation by lysosome inhibitors. HeLa cells were co-transfected with the M2 plus Vector (−), MARCH8 ( + ), or W114A ( + ). Cells were treated with DMSO, lysosomal inhibitor chloroquine (CQ, 50 μm) or proteasome inhibitor MG132 (25 μm) (a), DMSO or a vacuolar H+ translocating ATPase inhibitor (Balf A1, 100 nM) (b), followed by western blot. c HeLa cells co-transfected with M2 and MARCH8 plasmid were treated with DMSO or CQ and processed for immunofluorescence staining with anti-M2 (green) and anti-MARCH8 antibodies (red). Scale bars represent 10 μm. d, e HeLa cells transfected with EGFP fused M2 and vector, MARCH8 or W114A were permeabilized, then co-stained with MARCH8 (white) and EEA1 (red) antibodies (d) or with MARCH8 (white) and LAMP1 (red) antibodies (e). Scale bars represent 10 μm. f HeLa cells were co-transfected with M2 plus Vector, MARCH8 or W114A. The lyso-Tracker (red) was added to cells 30 min before fixation. M2 (green) and MARCH8 (white) were detected by immunostaining. Scale bars represent 10 µm. Pearson’s correlation coefficient analysis was based on multiple sight fields each group (n = 6 fields). *P < 0.01; **P < 0.001; n.s., nonsignificant, unpaired two-tailed Student t-test. g Co-immunoprecipitation of M2 and Flag-MARCH8 in HEK293T cells. h, i MARCH8 ubiquitinates IAV M2. HEK293T cells were transfected with HA-Ub, M2 and Vector, MARCH8 or W114A (h). HEK293T cells transfected with HA-Ub and Vector, MARCH8 or W114A were infected with WSN virus (MOI = 2) for 12 h (i). Whole-cell lysates were subjected to IP with anti-M2 antibody, and the IP and input were analyzed by western blotting with antibodies against the indicated targets.