Journal: Nature Communications
Article Title: Lysosome-targeting live attenuated influenza vaccines elicit robust and broad immunity in mice
doi: 10.1038/s41467-026-69920-0
Figure Lengend Snippet: a LTM-dependent degradation of viral NS1 protein. Western blot analysis shows reduced NS1 protein levels in cells expressing NS1-N LTM compared with the N LTM mutant , while NS1 mRNA levels remain comparable ( n = 3). b Lysosome dependence of LTM-mediated NS1 degradation. NS1-N LTM protein degradation is blocked by lysosomal inhibitors but not by proteasome or autophagy inhibition. HEK293T cells expressing either NS1-N LTM (left) or NS1-N LTM mutant (right) protein were cultured with or without the proteasome inhibitor MG-132 (10 µM), the autophagy inhibitor 3-methyladenine (3-MA; 10 mM), bafilomycin A1 (Baf A1; 0.4 µM), or chloroquine (CQ; 50 µM) for 6 h. The viral NS1 protein was detected by Western blotting ( n = 3). c Co-immunoprecipitation demonstrating interaction of HSC70 with NS1-N LTM but not with the NS1 LTM mutant ( n = 3). d Dependence of LTM-mediated NS1 degradation on LAMP2A. Conventional HEK293T cells and LAMP2A-KO HEK293T cells were transfected with constructs expressing NS1-N LTM (left) or NS1-N LTM mutant (right) protein and collected 24 h post-transfection. Viral NS1 protein was detected by Western blotting ( n = 3). Immunofluorescence analysis showing colocalization of NS1-N LTM with HSC70 ( e ) and LAMP2A ( f ), but not of the NS1-N LTM mutant . Green, NS1; red, HSC70 or LAMP2A; blue, nuclei; yellow, colocalization sites; scale bar, 5 µm. Representative images of at least three independent experiments are shown. LAMP2A-dependent degradation of NS1-N LTM during viral infection in conventional and LAMP2A-KO HEK293T ( g ) and A549 ( h ) cells. Replication competence of NS1-N LTM and NS1-N LTM mutant viruses in conventional and LAMP2A-KO HEK293T ( i ) and A549 ( j ) cells. Immunofluorescence staining of influenza viral M1 protein at 48 h after infection (MOI = 0.01) showing the replication competence of NS1-N LTM or NS1-N LTM mutant virus in conventional and LAMP2A-KO cells. Green, M1; blue, nuclei; scale bar, 100 µm. Viral titers in culture supernatants were quantified by immunofluorescence focus-forming unit (FFU) assay ( n = 3). Data are means ± s.d; n = 3 biologically independent experiments; unpaired two-tailed t -test for ( i ) and ( j ); *** P < 0.001. Source data are provided as a Source Data file.
Article Snippet: After another 6 h of culture, the cells were washed with PBS, fixed with 4% paraformaldehyde (PFA) for 20 min at room temperature, washed with PBS, permeabilized with PBS containing 0.1% Triton X-100 for 5 min, blocked with PBS containing with 0.1% Triton X-100 and 10% goat serum for 1 h at room temperature, and incubated with anti-Flag antibody (MBL, Cat# PM020; 1:1000 dilution) and anti-HSC70 antibody (Santa Cruz Biotechnology, Cat# sc-7298; 1:300 dilution) or anti-LAMP2A antibody (Santa Cruz Biotechnology, Cat# sc-18822; 1:300 dilution) diluted in PBS containing 0.1% Triton X-100 and 1% goat serum overnight at 4 °C, followed by incubation with Alexa Fluor 488 goat anti-rabbit IgG (Life Technologies, Cat# A11034; 1:1000 dilution) and Alexa Fluor 555 goat anti-mouse IgG (Life Technologies, Cat# A32727; 1:1000 dilution) for 1 h at room temperature.
Techniques: Western Blot, Expressing, Mutagenesis, Inhibition, Cell Culture, Immunoprecipitation, Transfection, Construct, Immunofluorescence, Infection, Staining, Virus, Two Tailed Test
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