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

Fig 3. Minimal viral partners and M1 basic residues essential for influenza A(H1N1)pdm09 M1 membrane localization.

Fig 3

HEK 293T cells were transfected with empty vector (mock) or with the pcDNA-M1 (M1 WT or mutant), pcDNA-M2 (M2), pHW2000-NS (NS1/NEP) or pHW2000-M (M) plasmids, as indicated. Cell fractionation experiments were performed 48h post-transfection. (A) Percentage of M1 detected in the nuclear, cell membrane or cytosolic fraction in each condition. M2-mut2 was used as control for low M1 membrane binding. The histograms show the result of at least three independent experiments (mean± standard deviation represented in the error bars). Differences between conditions were assessed using the Student’s t-test. (B) Cell fractionation controls. Fractions of cells co-expressing M1+M2+NS1/NEP were immunoblotted with antibodies against a membrane marker (LAMP2) and a cytosolic marker (the ribosomal S6 protein). Tubulin was used as loading control. PNS, Post-Nuclear Supernatant. (C) Expression of the indicated influenza A(H1N1)pdm09 viral proteins was checked after transfection in HEK 293T cells of the relevant plasmids by western blotting with anti-M1 (H1N1), anti-NEP and anti-NS1 antibodies. HA was detected with a serum obtained using an influenza A(H1N1)pdm09 strain isolated from a vaccinated patient.