Fig. 6. MARCH8 mediates K63-linked polyubiquitination of M2 at position K78.

a Illustration of IAV M2 domain-organization and the cytoplasmic tail amino-acid sequence of WSN virus M2 and the indicated mutants. Lysine residues (K) are marked as red, and the K > R mutants are marked as blue. b, c HEK293T cells were transfected with M2 mutants and vector or MARCH8. Surface M2 was determined by flow cytometry (b). Quantitation of M2 positive cells was shown in (c). Data shown are the means ± SD (n = 3 biological replicates). **P < 0.001; n.s., nonsignificant, unpaired two-tailed Student t-test, without any adjustments for multiple comparisons. d Ubiquitination of M2 mutants by MARCH8. HEK293T cells were transfected with M2 mutants and vector or MARCH8. Cell lysates were subject to IP with anti-M2 antibody, and the IP and input were analyzed by western blotting with antibodies against the indicated targets. e HeLa cells were co-transfected with WT or the K78R mutant M2 and vector or MARCH8. M2 and MARCH8 were detected with specific antibodies in confocal microscopy. Scale bars represent 10 µm. f HEK293T cells were transfected with HA-tagged ubiquitin mutants, M2 and MARCH8. Cell lysates were subject to IP with anti-M2 antibody, and the IP and input were analyzed by western blot with antibodies against the indicated targets.