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a A549 cells were transfected with siNC or siRNAs targeting Rab3b (siRab3b-1, siRab3b-2), Rab6a (siRab6a-1, siRab6a-2), Rab11a (siRab11a-1, siRab11a-2), Rab17 (siRab17-1, siRab17-2), Rab23a (siRab23a-1, siRab23a-2), Rab25 (siRab25-1, siRab25-2), Rab27a (siRab27a-1, siRab27a-2), Rab37 (siRab37-1, siRab37-2), or Rab38 (siRab38-1, siRab38-2) for 24 h. The cells were then infected with HM virus (MOI, 10), and HA protein levels on the cell surface were quantified by flow cytometry at 4 hpi. b A549 cells transfected with siNC or siRab27a (siRab27a-1, siRab27a-2) for 24 h were infected with HM virus (MOI, 0.1). c–e Rab27a KO and A549-Cas9 cells were infected with HM virus (MOI, 0.1), SH13/H9N2 virus (MOI, 0.01), or PR8/H1N1 virus (MOI, 0.01). f–h A549 cells were transfected with 2 μg/ml exogenous Rab27a or an empty vector as a negative control for 24 h. The cells were then infected with HM virus (MOI, 0.1), SH13 virus (MOI, 0.01), or PR8 virus (MOI, 0.01). i A549-Cas9, Rab27a KO, and Rab27a KO cells stably expressing Flag-Rab27a-WT, Flag-Rab27a-T23N (dominant-negative mutant), or Flag-Rab27a-Q78L (constitutively active mutant) were infected with HM virus (MOI, 0.1). Rab27a and NP protein expression levels were examined by western blot ( b , f , g , h , and i ), with GAPDH or β-actin serving as a loading control. Viral titers in the supernatants were quantified using a TCID 50 assay on MDCK cells ( b–i ). Data represent the means ± SD from three independent experiments. Statistical significance was determined using a two-tailed Student’s t -test.

Journal: Nature Communications

Article Title: Rab27a regulates the transport of influenza virus membrane proteins to the plasma membrane

doi: 10.1038/s41467-025-61587-3

Figure Lengend Snippet: a A549 cells were transfected with siNC or siRNAs targeting Rab3b (siRab3b-1, siRab3b-2), Rab6a (siRab6a-1, siRab6a-2), Rab11a (siRab11a-1, siRab11a-2), Rab17 (siRab17-1, siRab17-2), Rab23a (siRab23a-1, siRab23a-2), Rab25 (siRab25-1, siRab25-2), Rab27a (siRab27a-1, siRab27a-2), Rab37 (siRab37-1, siRab37-2), or Rab38 (siRab38-1, siRab38-2) for 24 h. The cells were then infected with HM virus (MOI, 10), and HA protein levels on the cell surface were quantified by flow cytometry at 4 hpi. b A549 cells transfected with siNC or siRab27a (siRab27a-1, siRab27a-2) for 24 h were infected with HM virus (MOI, 0.1). c–e Rab27a KO and A549-Cas9 cells were infected with HM virus (MOI, 0.1), SH13/H9N2 virus (MOI, 0.01), or PR8/H1N1 virus (MOI, 0.01). f–h A549 cells were transfected with 2 μg/ml exogenous Rab27a or an empty vector as a negative control for 24 h. The cells were then infected with HM virus (MOI, 0.1), SH13 virus (MOI, 0.01), or PR8 virus (MOI, 0.01). i A549-Cas9, Rab27a KO, and Rab27a KO cells stably expressing Flag-Rab27a-WT, Flag-Rab27a-T23N (dominant-negative mutant), or Flag-Rab27a-Q78L (constitutively active mutant) were infected with HM virus (MOI, 0.1). Rab27a and NP protein expression levels were examined by western blot ( b , f , g , h , and i ), with GAPDH or β-actin serving as a loading control. Viral titers in the supernatants were quantified using a TCID 50 assay on MDCK cells ( b–i ). Data represent the means ± SD from three independent experiments. Statistical significance was determined using a two-tailed Student’s t -test.

Article Snippet: The antibodies used in this study and their sources are as follows: Rabbit polyclonal antibodies: Rab37, Rab3b, Rab6a, Rab11a, Rab17, Rab23, Rab25, Rab27a, Rab27b, SYTL4, SQSTM1, ATB4B, TNG46, GM130 (Proteintech; 13051-1-AP, 15774-1-AP, 10187-2-AP, 20229-1-AP, 17501-1-AP, 11101-1-AP, 13189-1-AP, 16868-1-AP, 13412-1-AP, 12128-1-AP, 18420-1-AP, 15131-1-AP, 13573-1-AP, 11308-1-AP), Rab38 (Abways, AY3825), SYTL1 (Bethyl, A305-648A-T), BECN1 (ABclonal Biotechnology, A7353), Phospho-ATG4B (Ser316) (Affinity Biosciences, AF3505), ATG16L1 (HUABIO, ET7106-65), and IAV M2, NP, NS1, and HA (GeneTex; GTX125951, GTX30852, GTX125990, GTX127357).

Techniques: Transfection, Infection, Virus, Flow Cytometry, Plasmid Preparation, Negative Control, Stable Transfection, Expressing, Dominant Negative Mutation, Mutagenesis, Western Blot, Control, Two Tailed Test