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. 2016 Nov 4;11(11):e0165421. doi: 10.1371/journal.pone.0165421

Fig 4. Minimal viral partners and M1 basic residues essential for A(H1N1)pdm09 M1 membrane attachment using cell membrane flotation assay.

Fig 4

HEK 293T cells were transfected with empty vector (mock) or with pcDNA-M1 (WT or mutants), pcDNA-M2, pHW2000-NS, pHW2000-M, pHW2000-NP, -HA, or–NA, as indicated. M2-mut2, an M2 CT mutant was used as control for low M1 membrane binding. M*, M harboring the relevant mutations in M1 or M2 coding sequences. (A) Membrane flotation assays were performed as described in Methods. (B) LAMP-2 and S6 were used as, respectively, membrane and cytosolic fraction markers. Expression of the fusion protein M2-GFP (localized in the membrane fraction) was detected with an anti-GFP antibody. (C) Analysis by membrane flotation assays of the effect of M1 basic motif mutations on M1 membrane attachment. (D) Membrane flotation assays performed in the presence also of NP, HA and NA. The percentages of membrane-bound M1 are the mean ± standard deviation of three independent experiments (except for M1R77A and R77/78). The p values indicate significant differences relative to the minimal system. Mb, membrane; Cyto, cytosolic.