human embryonic kidney 293t cells  (ATCC)

Journal: Neurobiology of Stress

Article Title: USP11 drives stress-induced synaptic structural deficits and depression-like behaviors through GSK3β/mTOR signaling

doi: 10.1016/j.ynstr.2026.100791

Figure Lengend Snippet: USP11 directly interacts with GSK3β. (A) Volcano plot of proteins detected after USP11 immunoprecipitation from mouse mPFC. Log2 fold change (x-axis) shows enrichment versus control; log2 intensity (y-axis) reflects normalized quantitation in experimental samples. USP11 served as bait; GSK3β is highlighted as an interactor (log2 intensity USP11 = 22.9, log2FC = 2.38). (B) Immunoprecipitation (IP) with anti-USP11 antibody, immunoblot (IB) detection for USP11 (110 kDa) and GSK3β (47 kDa). IP with anti-GSK3β or anti-USP11 antibody. Input: whole lysate; IgG: isotype control. (C) Validation in HEK293T transfection system: lysates of vector control or Flag-USP11 transfected cells (Flag tag, 110 kDa) subjected to IP (anti-GSK3β), IB for anti-USP11. (D) Cell lysate analysis of HEK293T single His-GSK3β, single Flag-USP11, or co-transfected groups, immunoblotted for His-GSK3β (47 kDa) and Flag-USP11 (110 kDa). (E, F) Reciprocal Co-IP verification from HEK293T co-transfection. Immunoblot analysis for His and Flag tag in His-GSK3β, Flag-USP11, and co-transfected samples. (E) Lane 1: His-GSK3β group (IP-His), Lane 2: Flag-USP11 group (IP-Flag), Lane 3: Co-transfection (IP- His) (F) Lane 1: His -GSK3β group (IP- His), Lane 2: Flag-USP11 group (IP-Flag), Lane 3: Co-transfection (IP-Flag). (G) Dot blot analysis showing specific binding between USP11 and GSK3β. BSA (100/200/500 ng) served as negative control, and purified USP11 (100/200/500 ng) was spotted on the same nitrocellulose membrane. After incubation with GSK3β protein solution, binding was detected by fluorescence imaging. (H) Immunofluorescence analysis of co-localization: Exogenous expression in HEK293T cells demonstrates USP11 (red) and GSK3β (green); endogenous expression verified in primary neurons. Nuclei stained with DAPI (blue), scale bar = 25 μm. (I) Fluorescence intensity profiles along linear ROIs: Gray values of USP11 (red) and GSK3β (green) measured with ImageJ. Dual-channel curves plotted in GraphPad Prism using exported data. (J) Pearson's correlation scatter plots for USP11(red) and GSK3β(green) fluorescence, generated using ScatterJ plugin for ImageJ. Pearson's r value shown. (K) Schematic of Flag-tagged USP11 fragment constructs used for pulldown mapping. (L) HEK293T cells were co-transfected with Flag-USP11 or its deletion mutant and His- GSK3β, followed by immunoprecipitation and immunoblot analysis for Flag and His. (M) Computational molecular docking predicts multiple direct contact sites between USP11 and GSK3β.

Article Snippet: Human embryonic kidney 293T (HEK293T) cells were obtained from Procell Life Science & Technology Co., Ltd. (Wuhan, China).

Techniques: Immunoprecipitation, Control, Quantitation Assay, Western Blot, Biomarker Discovery, Transfection, Plasmid Preparation, FLAG-tag, Co-Immunoprecipitation Assay, Cotransfection, Dot Blot, Binding Assay, Negative Control, Purification, Membrane, Incubation, Fluorescence, Imaging, Immunofluorescence, Expressing, Staining, Generated, Construct, Mutagenesis

Journal: Neurobiology of Stress

Article Title: USP11 drives stress-induced synaptic structural deficits and depression-like behaviors through GSK3β/mTOR signaling

doi: 10.1016/j.ynstr.2026.100791

Figure Lengend Snippet: USP11 regulates GSK3β ubiquitination, phosphorylation, and synaptic protein homeostasis in neural cells (A) Western blot analysis of GSK3β ubiquitination in HEK293T cells co-transfected with Flag-vector (control), Flag-USP11 (wild-type, 110 kDa), or Flag-USP11-C318S (catalytically inactive mutant). Endogenous GSK3β and phosphorylated GSK3β at Ser9 were immunoprecipitated from cell lysates using anti-GSK3β antibody, and ubiquitination levels were detected by immunoblotting with anti-ubiquitin antibody. GSK3β: 47 kDa; ubiquitin bands detected as smear. (B) Western blot analysis of GSK3β phosphorylation in three 293T cell groups: wild-type (Ctrl), stable USP11-overexpressing line generated by lentiviral transduction (USP11-OE), and USP11-overexpressing cells subjected to siRNA knockdown (USP11-OE + siUSP11). siUSP11 was transfected to silence USP11 in the stable overexpressing cell line. Whole cell lysates were analyzed for endogenous USP11 (110 kDa), phosphorylated GSK3β at Ser9 (p-GSK3β, 47 kDa), total GSK3β (47 kDa), and GAPDH (35 kDa) as loading control. Representative results from n = 3 biological replicates per group. (C) Gray value quantification of p-GSK3β/t-GSK3β in 293T cells (n = 3, F (2, 6) = 35.38, p = 0.0005). (D) Western blot analysis of USP11 (110 kDa), phosphorylated mTOR (p-mTOR, Ser2448, 289 kDa), total mTOR (289 kDa), p-GSK3β (Ser9, 47 kDa), total GSK3β (47 kDa), and Tubulin (55 kDa) in primary neurons upon USP11 siRNA knockdown (n = 3). (E, F) Gray value quantification of p-GSK3β/t-GSK3β, and p-mTOR/t-mTOR ratios in neurons upon USP11 siRNA knockdown (n = 3, p-GSK3β, p = 0.0213, p-mTOR, p = 0.0047). (G) Immunoblot of USP11 (110 kDa), p-GSK3β (Ser9, 47 kDa), total GSK3β (47 kDa), SYN (77 kDa), and Tubulin (55 kDa) in primary neurons infected with adeno-associated virus (AAV) (n = 3). (H, I) Gray value quantification of p-GSK3β/t-GSK3β, and SYN/Tubulin ratios in neurons transduced with vector or AAV-USP11 viruses (n = 3, p-GSK3β, p = 0.0078, SYN, Welch's t -test, p = 0.0031). (J) Representative immunofluorescence of primary neurons transduced with vector or AAV-USP11 viruses, showing DAPI (blue, nuclei), SYN (green, synaptophysin), and USP11 (magenta); merged panels display synapse integrity. Scale bar: 50 μm. (K, L) Quantitative analysis from three independent biological replicates in primary neurons transduced with vector or AAV-USP11 viruses (K) Mean USP11 immunofluorescence intensity (p = 0.0416), (L) Mean SYN immunofluorescence intensity (p = 0.0035). Data are shown as mean ± SEM. Determined by t -test (baseline comparisons) or one-way ANOVA (multiple groups) unless otherwise indicated. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Article Snippet: Human embryonic kidney 293T (HEK293T) cells were obtained from Procell Life Science & Technology Co., Ltd. (Wuhan, China).

Techniques: Ubiquitin Proteomics, Phospho-proteomics, Western Blot, Transfection, Plasmid Preparation, Control, Mutagenesis, Immunoprecipitation, Generated, Transduction, Knockdown, Infection, Virus, Immunofluorescence

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