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: Puromycin (HY-B1743), LY294002 (HY-10108), and MG132 (HY-13259) were purchased from MedChemExpress.

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