Journal: Signal Transduction and Targeted Therapy

Article Title: Investigating the effects and underlying mechanisms of glycosylation sites on the immunogenicity of COVID-19 vaccines

doi: 10.1038/s41392-026-02751-z

Figure Lengend Snippet: Influence of glycosylation site mutations on antigenicity. a Activation of MHC-I- and MHC-II-expressing immune cells; b Binding ability of the antigen to antibodies and the ACE2 receptor: when the RBD was used as the standard (white), if the affinity was greater than that of the RBD, the color was blue; if the affinity was less than that of the RBD, the color was red. The deeper the color was, the stronger (blue) or weaker (red) the degree of antigen binding. The data were presented as the mean ± SD, and each symbol represented a mouse ( n = 3). Statistical analysis was conducted using one-way ANOVA and Tukey’s multiple comparison tests for bar graphs; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; n.s. not significant

Article Snippet: For the 11 monoclonal antibodies, horseradish peroxidase (HRP-anti-mouse-IgG ABclonal, AS003): PBS was diluted at a ratio of 1:5000; for ACE2, HRP-anti-human-IgG (ABclonal, AS003): PBS was diluted at a ratio of 1:5000; 50 μL was added to each well.

Techniques: Glycoproteomics, Activation Assay, Expressing, Binding Assay, Comparison

Journal: mBio

Article Title: Secretory full-length human prosaposin (PSAP) inhibits SARS-CoV-2 infection through facilitating the release of S1 subunit of spike protein

doi: 10.1128/mbio.00129-26

Figure Lengend Snippet: PSAP inhibits SARS-CoV-2 pseudovirus infection. ( A ) Identification of candidate genes restricting viral entry. The scatter plot shows Z -scores from the cDNA overexpression screen, ranked by Z -score. The dotted lines indicate the cutoff values. Genes with significantly different Z -score are highlighted. ( B ) Validation of top hits in a pseudovirus entry assay. 293T-ACE2 cells were transfected with 1 µg control vector (pCMV6-GST-Flag) or increasing doses (0.2, 0.5, 0.7, and 1 µg) of plasmids encoding FUBP1, NSD2, IFNA8, IFNA2, GZMB, and PSAP. At 36 h post-transfection, cells were then challenged with SARS-CoV-2 pseudovirus at a 4 × 10 7 TU for 24 h. Expression levels of GFP, ACE2, Flag, and β-actin were analyzed by immunoblotting with specific antibodies.

Article Snippet: Additional antibodies for ACE2 (A12737), β-actin (AC026) were purchased from ABclonal.

Techniques: Infection, Over Expression, Biomarker Discovery, Transfection, Control, Plasmid Preparation, Expressing, Western Blot

Journal: mBio

Article Title: Secretory full-length human prosaposin (PSAP) inhibits SARS-CoV-2 infection through facilitating the release of S1 subunit of spike protein

doi: 10.1128/mbio.00129-26

Figure Lengend Snippet: The secreted extracellular PSAP inhibits SARS-CoV-2 pseudovirus infection. ( A ) 293T-ACE2 cells were transfected with control siRNA or three independent PSAP-targeting siRNA (#1, 2, and 3) for 48 h. Cells were then challenged with SARS-CoV-2 pseudovirus. Cell lysates were analyzed by Western blotting using antibodies against PSAP, GFP, and β-actin. ( B ) Validation of PSAP knockout clones confirms its restriction activity. 293T-ACE2 wild-type and PSAP knockout cells were infected with SARS-CoV-2 pseudovirus, and lysates were analyzed by Western blotting using the indicated antibodies. ( C–F ) 293T-ACE2 cells were transfected with 1 μg of control vector (pCMV6-GST-Flag) or increasing doses (0.2, 0.5, 0.7, and 1 μg) of plasmids encoding full-length PSAP-Flag ( C ) or a signal peptide-deleted mutant (PSAP-delSP-Flag) ( E ). At 36 h post-transfection, cell lysates and culture supernatants were anzlyzed by Western blot using the indicated antibodies. Supernatants collected from the transfection in panels C and E were pre-incubated with SARS-CoV-2 pseudoviruses at 37°C for 1 h. The mixtures were then used to infect 293T-ACE2 cells respectively ( D and F ). Cell lysates were analyzed by Western blotting using the indicated antibodies. ( G and H ) Indicated concentrations of PSAP-His protein (100, 20, 4, 0.8, 0.16, 0.032, and 0 μg/mL) were incubated with SARS-CoV-2 pseudovirus or VSV-G-pseudotyped lentivirus at 37°C for 1 h. The mixtures were then used to infect 293T-ACE2 cells. Cell lysates were analyzed by Western blotting using the indicated antibodies.

Article Snippet: Additional antibodies for ACE2 (A12737), β-actin (AC026) were purchased from ABclonal.

Techniques: Infection, Transfection, Control, Western Blot, Biomarker Discovery, Knock-Out, Clone Assay, Activity Assay, Plasmid Preparation, Mutagenesis, Incubation

Journal: mBio

Article Title: Secretory full-length human prosaposin (PSAP) inhibits SARS-CoV-2 infection through facilitating the release of S1 subunit of spike protein

doi: 10.1128/mbio.00129-26

Figure Lengend Snippet: PSAP inhibits SARS-CoV-2 infection. ( A ) 293T-ACE2 cells were transfected with 1 μg of control vector (pCMV6-GST-Flag) and increasing doses of PSAP-Flag (0.2, 0.5, 0.7, and 1 μg) for 36 h and then challenged with SARS-CoV-2 authentic virus at an MOI of 0.05 for 24 h. Cells were then harvested, and Western blotting was performed using the indicated antibodies. ( B ) 293T-ACE2 wild-type or PSAP knockout cells were infected with SARS-CoV-2 at an MOI of 0.05 for 24 h. Viral N gene expression was measured by RT-qPCR. ( C ) Indicated concentrations of PSAP-His (0, 0.032, 0.16, 0.8, 4, 20, and 100 μg/mL) were incubated with SARS-CoV-2 at 37°C for 1 h, and then 293T-ACE2 cells were infected. Twenty-four hours later, cells were harvested, and viral N gene expression was quantified by RT-qPCR. ( D ) Indicated concentrations of PSAP-His protein (0,0.8, 4,20,and 100 μg/mL) were incubated with SARS-CoV-2. The mixtures were used to infect 293T-ACE2, Calu-3, and Huh7 cells. Viral N gene expression was quantified by RT-qPCR. ( E ) 293T-ACE2 cells were infected with different MOIs of SARS-CoV-2, and the cell supernatant and cell pellets were collected for Western blotting. ( F ) A time-of-addition assay with PSAP was performed. 293T-ACE2 cells were treated with PSAP at different stages: pre-treatment, during viral incubation, or post-entry of SARS-CoV-2 at an MOI of 0.05. The inhibitory effect was assessed by quantifying intracellular vRNA at 12 h post-infection. These experiments were repeated at least twice. *** P < 0.001; ** P < 0.01; * P < 0.05; ns, P > 0.05.

Article Snippet: Additional antibodies for ACE2 (A12737), β-actin (AC026) were purchased from ABclonal.

Techniques: Infection, Transfection, Control, Plasmid Preparation, Virus, Western Blot, Knock-Out, Gene Expression, Quantitative RT-PCR, Incubation

Journal: mBio

Article Title: Secretory full-length human prosaposin (PSAP) inhibits SARS-CoV-2 infection through facilitating the release of S1 subunit of spike protein

doi: 10.1128/mbio.00129-26

Figure Lengend Snippet: PSAP interacts with spike protein through the RBD. ( A–F ) HEK293T cells were co-transfected with plasmids expressing PSAP-Flag and spike-HA ( A ), S1-HA ( B ), S1-delRBD-HA ( C ), S1-delNTD-HA ( D ), spike-delRBD ( E ), or RBD-HA ( F ). After 36 h, cells were harvested, and lysates were subjected to immunoprecipitation with anti-Flag antibody, followed by Western blotting using the indicated antibodies. PSAP-only transfection and control mouse IgG were used as negative as control. ( G, H ) Binding of PSAP or ACE2 to SARS-CoV-2 S1 was measured by bio-layer interferometry (BLI). Serial twofold dilutions of each analyte were used for the binding assays.

Article Snippet: Additional antibodies for ACE2 (A12737), β-actin (AC026) were purchased from ABclonal.

Techniques: Transfection, Expressing, Immunoprecipitation, Western Blot, Control, Binding Assay

Journal: mBio

Article Title: Secretory full-length human prosaposin (PSAP) inhibits SARS-CoV-2 infection through facilitating the release of S1 subunit of spike protein

doi: 10.1128/mbio.00129-26

Figure Lengend Snippet: Molecular docking simulation of the PSAP-S1 interaction. Predicted binding model of PSAP (red) with the S1 subunit (blue) generated by molecular docking. Green shading on the S1 highlights residues predicted to form intermolecular contacts with PSAP. For comparison, residues on the S1 structure predicted to form contacts with ACE2 are highlighted in purple.

Article Snippet: Additional antibodies for ACE2 (A12737), β-actin (AC026) were purchased from ABclonal.

Techniques: Binding Assay, Generated, Comparison

Journal: mBio

Article Title: Secretory full-length human prosaposin (PSAP) inhibits SARS-CoV-2 infection through facilitating the release of S1 subunit of spike protein

doi: 10.1128/mbio.00129-26

Figure Lengend Snippet: PSAP facilitates the release of the SARS-CoV-2 S1 subunit. ( A ) An in vitro ELISA competition experiment was performed. The coated S1-HA was incubated with PSAP-His protein, GST protein, or ACE2-mFc in different concentrations. The signal was detected by HRP-anti-mouse secondary antibody and TMB enzymatic reaction. ( B ) 293T cells stably expressing S protein were incubated with PSAP-GST or GST protein for the indicated times. Cell surface S1 subunit was detected by flow cytometry using an anti-RBD antibody. The mean fluorescence intensity of the S1 subunit on the cell membrane was shown. ( C and D ) 293T cells stably expressing the SARS-CoV-2 S protein ( C ) or S-D614G ( D ) were incubated with increasing doses of PSAP-His protein. Cell culture supernatants and cell lysates (pellet) were collected and analyzed by Western blotting to detect S1 subunit levels. These experiments were repeated at least twice. *** P < 0.001; ** P < 0.01; * P < 0.05; ns, P > 0.05.

Article Snippet: Additional antibodies for ACE2 (A12737), β-actin (AC026) were purchased from ABclonal.

Techniques: In Vitro, Enzyme-linked Immunosorbent Assay, Incubation, Stable Transfection, Expressing, Flow Cytometry, Fluorescence, Membrane, Cell Culture, Western Blot