Journal: bioRxiv
Article Title: c-Fos enhances influenza virus replication by stabilizing the M2 protein and promoting autophagosome accumulation
doi: 10.64898/2026.03.05.709812
Figure Lengend Snippet: (A) Cell viability was assessed by CCK-8 assay following T5224 treatment. For infection experiments, cells were pretreated with 0, 10, or 20 μM T5224 for 2 h before PR8 virus infection. Protein lysates were harvested at 24 h post-infection and analyzed by Western blot using anti-NP and anti-GAPDH antibodies. (B) 293H cells were co-transfection with M2 and c-Fos plasmids, alongside M2 and vector controls. After 48 h, cells were lysed and RNA was extracted for RT-qPCR analysis of M2 mRNA expression. (C and D) Co-immunoprecipitation and immunoblot analysis of 293H cells co-transfected with Flag-c-Fos and M2 (C) or NP (D). (E) 293H cells were co-transfection with Flag-c-Fos and M2 for 48 h, followed by immunostaining for Flag (red) and M2 (green). Representative confocal immunofluorescence images are shown. Scale bars, 2 μm. (F) 293H cells were co-transfected with M2 and Flag-c-Fos or empty vector. Cells were treated with 50 μg/mL CHX and harvested at the indicated time points (0 - 8 h). M2 protein levels were analyzed by Western blot. (G and H) 293H cells were transfected with M2 or empty vector. At 6 h post-transfection, cells were treated with MG132 (100, 200, 400 nM), Bafilomycin A1 (1, 2, 5 nM), or DMSO control. After 48 h, M2 protein expression was assessed by Western blot. (I) 293H cells were co-transfected with M2 and Flag-c-Fos or vector, followed by treatment with MG132 (200 nM) or Bafilomycin A1 (2 nM) for 48 h. M2 protein levels were analyzed by Western blot. All data are presented as means ± SD from at least three independent experiments. Significance was determined by Student’s t test: *, P < 0.05; **, P < 0.01; ***, P < 0.001. #, P > 0.05.
Article Snippet: The following primary antibodies were used: anti-LC3B (2775; Cell Signaling Technology, CST), anti-SQSTM1 (5114; CST), anti-GAPDH (2118; CST), anti-NP (10780-01; SouthernBiotech), anti-SERCA (A1097; ABclonal), anti-Flag (AE092; ABclonal), and anti-influenza A virus M2 (IT-003-015; Immune Technology).
Techniques: CCK-8 Assay, Infection, Virus, Western Blot, Cotransfection, Plasmid Preparation, Quantitative RT-PCR, Expressing, Immunoprecipitation, Transfection, Immunostaining, Immunofluorescence, Control
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