Journal: Signal Transduction and Targeted Therapy

Article Title: A nanobody-based proteolysis-targeting chimera offers broad-spectrum protection against diverse influenza virus infections

doi: 10.1038/s41392-026-02666-9

Figure Lengend Snippet: Design and validation of Nb-PROTACs targeting the influenza NP protein. a Schematic representation of the Nb-PROTAC constructs. b NP-eGFP degradation monitored by fluorescence microscopy. We cotransfected 293T cells with plasmids encoding individual Nbs or VHL-Nbs together with NP-eGFP, followed by incubation for 48 h. Fluorescence images were captured via an inverted fluorescence microscope equipped with a 10 × objective. c Western blot analysis of NP degradation. We cotransfected 293T cells with plasmids expressing the indicated Nb or VHL-Nb constructs together with PR8-NP. The cell lysates were harvested and subjected to Western blot analysis. The blot shown is representative of three independent experiments performed. Densitometric quantification of NPs normalized to GAPDH (NP/GAPDH ratio) is shown below the blot; the percentages on the bars indicate the NP abundance relative to that of the corresponding controls. d Viral replication kinetics in A549 cells transduced with lentiviral vectors expressing the indicated constructs. The data represent the means ± SDs of three independent experiments ( n = 3). Asterisks indicate statistically significant reductions in viral titers compared with those in the corresponding Nb group (* p < 0.05, ** p < 0.01, *** p < 0.001). Scale bar = 300 μm

Article Snippet: The human embryonic kidney cell line 293T, murine lung epithelial cell line MLE-12, and human lung epithelial cell line A549 were obtained from the American Type Culture Collection (ATCC).

Techniques: Biomarker Discovery, Construct, Fluorescence, Microscopy, Incubation, Western Blot, Expressing, Transduction

Journal: Signal Transduction and Targeted Therapy

Article Title: A nanobody-based proteolysis-targeting chimera offers broad-spectrum protection against diverse influenza virus infections

doi: 10.1038/s41392-026-02666-9

Figure Lengend Snippet: Binding analysis of Nb-PROTACs targeting NP proteins across 16 influenza A virus subtypes. Binding activities of VHL-Nb135 ( a ) and VHL-Nb170 ( b ) to NP proteins derived from 16 influenza virus subtypes (Supplementary Table ). We cotransfected 293T cells for 48 h with plasmids expressing individual NP proteins from each of the 16 influenza virus subtypes, along with plasmids encoding VHL-Nb135 ( a ) or VHL-Nb170 ( b ). Whole-cell lysates were prepared and immunoprecipitated via anti-Myc-tag antibody-conjugated agarose beads, followed by Western blotting with the indicated antibodies

Article Snippet: The human embryonic kidney cell line 293T, murine lung epithelial cell line MLE-12, and human lung epithelial cell line A549 were obtained from the American Type Culture Collection (ATCC).

Techniques: Binding Assay, Virus, Derivative Assay, Expressing, Immunoprecipitation, Western Blot

Journal: Signal Transduction and Targeted Therapy

Article Title: A nanobody-based proteolysis-targeting chimera offers broad-spectrum protection against diverse influenza virus infections

doi: 10.1038/s41392-026-02666-9

Figure Lengend Snippet: Broad-spectrum degradation activities of Nb-PROTACs targeting NP proteins across 16 influenza A virus subtypes. Degradation activities of VHL-Nb135 ( a ) and VHL-Nb170 ( b ) against NP proteins across 16 influenza virus subtypes. We cotransfected 293T cells for 36 h with plasmids expressing Nb135, VHL-Nb135, Nb170, or VHL-Nb170, along with individual NP-expressing plasmids from each influenza subtype. The cell lysates were harvested and analyzed via Western blotting with the indicated antibodies. The blot shown is representative of three independent experiments. Densitometric quantification of the NP protein levels normalized to the level of GAPDH is shown below the blot image. The data represent the means ± SDs from three independent Western blot experiments ( n = 3). Statistical significance was determined via one-way ANOVA followed by Tukey’s post hoc test (* p < 0.05, ** p < 0.01, *** p < 0.001)

Article Snippet: The human embryonic kidney cell line 293T, murine lung epithelial cell line MLE-12, and human lung epithelial cell line A549 were obtained from the American Type Culture Collection (ATCC).

Techniques: Virus, Expressing, Western Blot

Journal: Signal Transduction and Targeted Therapy

Article Title: A nanobody-based proteolysis-targeting chimera offers broad-spectrum protection against diverse influenza virus infections

doi: 10.1038/s41392-026-02666-9

Figure Lengend Snippet: Degradation mechanism of Nb-PROTAC. a VHL-Nb170 induces the ubiquitination of the NP protein. We transfected 293T cells with plasmids expressing Nb170 or VHL-Nb170 along with PR8-NP and HA-tagged ubiquitin (HA-Ub). At 48 h posttransfection, the cell lysates were subjected to coimmunoprecipitation with an anti-NP antibody conjugated to protein G agarose beads, followed by Western blotting with the indicated antibodies. b Degradation inhibition assay. We cotransfected 293T cells with plasmids expressing Nb170 or VHL-Nb170 along with PR8-NP for 24 h. The cells were then treated with MG132 (10 μM), LY294002 (20 μM), or DMSO (control) for an additional 8 h. Cell lysates were subsequently collected and analyzed by immunoblotting with the indicated antibodies. Confirmation of NP degradation through the autophagy pathway. Autophagy-related protein 5 knockout (293T- ATG5 -/- ) or wild-type 293T cells were transfected with pCAGGS-NP-GFP or pCAGGS-NP together with pCAGGS-Nb170 or pCAGGS-VHL-Nb170 for 36 h. c NP-eGFP expression was assessed via immunofluorescence microscopy. d NP expression was analyzed by immunoblotting with the indicated antibody. The blot shown is representative of three independent experiments. Densitometric quantification of the NP protein levels normalized to the level of GAPDH is shown below the blot image. e Identification of autophagy cargo receptors involved in NP degradation. 293T cells were transfected with plasmids encoding VHL-Nb170-Myc and PR8NP-His, along with plasmids expressing HA-tagged autophagy cargo receptors as indicated. After 36 h, the cell lysates were subjected to immunoprecipitation with anti-His antibody-conjugated protein G agarose. Both whole-cell lysates and immunoprecipitates were analyzed by immunoblotting with the indicated antibodies. Asterisks indicate significant differences (*** p < 0.001, ns > 0.05). Scale bar = 300 μm

Article Snippet: The human embryonic kidney cell line 293T, murine lung epithelial cell line MLE-12, and human lung epithelial cell line A549 were obtained from the American Type Culture Collection (ATCC).

Techniques: Ubiquitin Proteomics, Transfection, Expressing, Western Blot, Inhibition, Control, Knock-Out, Immunofluorescence, Microscopy, Immunoprecipitation