Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Enolase 1 suppresses influenza A virus replication by blocking the nuclear import of the viral ribonucleoprotein complex

doi: 10.1007/s00018-026-06146-9

Figure Lengend Snippet: ENO1 interacts with the NP protein. ( A , B ) HEK293T cells were transfected individually or in combination with Flag-tagged ENO1 and HA-tagged NP. At 24 h post-transfection, lysates of cells were immunoprecipitated with mouse anti-Flag mAb (A) or mouse anti-HA mAb (B) and then western blotted with rabbit anti-Flag pAb and rabbit anti-HA pAb, respectively. ( C ) HEK293T cells were infected with H1N1 virus (MOI = 5). At 12 h p.i., lysates from HEK293T cells were immunoprecipitated with mouse anti-ENO1 pAb, followed by western blotting with rabbit anti-ENO1 pAb and rabbit anti-NP pAb, respectively. ( D ) Purified GST and GST-ENO1 protein were used to pull down Flag-tagged NP. ( E ) A549 cells were uninfected or infected with H1N1 virus (MOI = 5). At 12 h p.i., the cells were fixed and stained with mouse anti-NP mAb and rabbit anti-ENO1 pAb. Scale bars, 5 μm. ( F , G ) HEK293T cells were transfected with the indicated plasmids. At 24 h post-transfection, cell lysates were immunoprecipitated with mouse anti-Flag mAb, followed by western blotting with rabbit anti-Flag pAb, rabbit anti-HA pAb, and rabbit anti-V5 pAb. The data are representative of at least three independent experiments

Article Snippet: The following primary antibodies were obtained from commercial sources: mouse anti-Flag mAb (A00187 -100, GenScript), rabbit anti-Flag pAb (20543-1-AP, Proteintech) mouse anti-HA mAb (A01244-100, GenScript), rabbit anti-HA pAb (51064-2-AP, Proteintech), mouse anti-V5 mAb (A01724-100, GenScript), rabbit anti-V5 pAb (14440-1-AP, Proteintech), rabbit anti-GAPDH pAb (10494-1-AP, Proteintech), rabbit anti-ENO1 pAb (11204-1-AP, Proteintech), rabbit anti-LaminA/C pAb (10298-1-AP, Proteintech), rabbit anti-KPNA4 pAb (12463-1-AP, Proteintech), rabbit anti-PB2 pAb (GTX125926, GeneTex), rabbit anti-PB1 pAb (GTX125923, GeneTex), rabbit anti-PA pAb (GTX118991, GeneTex), rabbit anti-NP pAb (GTX125989, GeneTex).

Techniques: Transfection, Immunoprecipitation, Western Blot, Infection, Virus, Purification, Staining

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Enolase 1 suppresses influenza A virus replication by blocking the nuclear import of the viral ribonucleoprotein complex

doi: 10.1007/s00018-026-06146-9

Figure Lengend Snippet: ENO1 impairs the nuclear import of the vRNP complex of IAV. ( A ) HEK293T cells were transfected with HA-tagged NP and Flag-tagged NP, together with Flag-tagged ENO1 or not. At 24 h post-transfection, cell lysates were immunoprecipitated with mouse anti-HA mAb and subjected to western blotting. ( B ) The effect of ENO1 on the vRNP assembly was determined using a co-IP assay. HEK293T cells were transfected with plasmids expressing vRNP proteins (PB2, PB1, PA, NP) and pPol I, together with Flag-tagged ENO1. Cell lysates were immunoprecipitated with mouse anti-HA antibody, followed by western blotting. ( C ) Si-ENO1- or NC-treated A549 cells were infected with H1N1 virus (MOI = 5). At 3 h p.i., the cells were separated into nuclear and cytoplasmic fractions, and each fraction was then western blotted with rabbit anti-ENO1, NP, GAPDH, and Lamin A/C pAb. The intensities of the NP protein bands were determined by using lmageJ, and the ratio of nuclear NP were normalized to cytoplasmic NP. ( D ) A549 cells transfected with Flag-tagged ENO1 or empty vector were infected with H1N1 virus (MOI = 5). At 3 h p.i., the cells were separated into nuclear and cytoplasmic fractions, and each fraction was then western blotted with rabbit anti-ENO1, NP, GAPDH, and Lamin A/C pAb. The intensities of the NP protein bands were determined by using lmageJ, and the ratio of nuclear NP were normalized to cytoplasmic NP. ( E ) A549 cells transfected with Flag-tagged ENO1 or empty vector were infected with H1N1 virus (MOI = 5). At 3 h p.i., the cells were fixed and stained with mouse anti-NP mAb and rabbit anti-ENO1 pAb. Scale bars, 20 μm. The data are representative of at least three independent experiments

Article Snippet: The following primary antibodies were obtained from commercial sources: mouse anti-Flag mAb (A00187 -100, GenScript), rabbit anti-Flag pAb (20543-1-AP, Proteintech) mouse anti-HA mAb (A01244-100, GenScript), rabbit anti-HA pAb (51064-2-AP, Proteintech), mouse anti-V5 mAb (A01724-100, GenScript), rabbit anti-V5 pAb (14440-1-AP, Proteintech), rabbit anti-GAPDH pAb (10494-1-AP, Proteintech), rabbit anti-ENO1 pAb (11204-1-AP, Proteintech), rabbit anti-LaminA/C pAb (10298-1-AP, Proteintech), rabbit anti-KPNA4 pAb (12463-1-AP, Proteintech), rabbit anti-PB2 pAb (GTX125926, GeneTex), rabbit anti-PB1 pAb (GTX125923, GeneTex), rabbit anti-PA pAb (GTX118991, GeneTex), rabbit anti-NP pAb (GTX125989, GeneTex).

Techniques: Transfection, Immunoprecipitation, Western Blot, Co-Immunoprecipitation Assay, Expressing, Infection, Virus, Plasmid Preparation, Staining