Journal: The Journal of Neuroscience

Article Title: Subunit-Dependent Surface Mobility and Localization of NMDA Receptors in Hippocampal Neurons Measured Using Nanobody Probes

doi: 10.1523/JNEUROSCI.2014-22.2023

Figure Lengend Snippet: Drift correction and localization error of QD probes in fixed hippocampal neurons expressing the YFP-tagged GluN1-1a subunit. A , Representative images of hippocampal neurons. Left, Neurons were stained by incubation in 20 n m MitoTracker Deep Red FM marker (catalog #M22426, Thermo Fisher Scientific) for 30 s in Neurobasal medium. Right, Neurons were infected with a lentivirus expressing the synaptic protein tdTomato-Homer1c. B , Schematic diagram depicting the three QD-based probes used in this study. The antiGFP-QD605 probe contains the rabbit anti-GFP IgG antibody combined with a secondary IgG antibody conjugated to QD605. The nanoGFP-QD605 probe contains an anti-GFP nanobody conjugated to QD605, while the nanoGFP-QD525 probe contains the anti-GFP nanobody conjugated to QD525. C , Negatively stained samples of both nanoGFP-QD probes were imaged at a magnification of 60,000× and a pixel size of 1.939 Å/px by TEM. Measured average diameters of both nanoGFP-QD probes ± SEM were 16.2 ± 0.4 nm (nanoGFP-QD525) and 20.4 ± 0.6 nm (nanoGFP-QD605; n ≥ 30). D , Example of drift estimated from multiple QD trajectories of the YFP-GluN1-1a subunit. E , The corresponding xy drift path. F , Example of raw QD trajectories (gray) and the drift-corrected QD trajectories (red) obtained by subtracting the drift path. G , Example images of fixed hippocampal neurons expressing the synaptic marker tdTomato-Homer1c (background pixels) and the YFP-GluN1-1a subunit labeled and tracked with the indicated QD-based probes (red). H , Scatter plots of all QD localizations; the red “+” indicates the mean value in both axes. I , Histograms showing the distribution of the distances between each QD localization shown in H ; the data were fitted with a Gaussian function, and the corresponding sigma (σ) values are indicated. J , Box plot summarizing the Gaussian fits performed on fixed QDs ( n = 20/group); one-way ANOVA F (2,57) = 121.84, p < 0.0001 followed by Bonferroni's multiple-comparisons test with p -values denoted in the figure. The average (mean ± SEM) localization errors were σ = 6.62 ± 0.25 nm (antiGFP-QD605), σ = 6.98 ± 0.32 nm (nanoGFP-QD605), and 15.34 ± 0.66 nm (nanoGFP-QD525).

Article Snippet: The bacterial expression vector encoding GFP nanobody (nanoGFP; catalog #49172, Addgene) was a gift from Brett Collins (The Institute for Molecular Bioscience, Queensland).

Techniques: Expressing, Staining, Incubation, Marker, Infection, Labeling

Journal: Pharmaceutics

Article Title: Biomimetic Cell-Derived Nanoparticles: Emerging Platforms for Cancer Immunotherapy

doi: 10.3390/pharmaceutics15071821

Figure Lengend Snippet: Application of biomimetic cell-derived nanoparticles in ACT.

Article Snippet: NK92MI cell membrane equipped with the anti-EGFR nanobody 7D12 , -- , -- , -- , EGFR positive cancer cell lines, including LoVo, MDA-MB-468, A549, and A431 cells, 7D12 for specifically recognizing EGFR positive cancer cells , The biomimetic delivery system exhibited high targeting for EGFR-positive cancer cells, caused enhanced secretion of cytotoxic effectors, and improved NK cell-based ACT. , [ ] .

Techniques: Membrane, Produced, Amplification, Activation Assay, Modification, Activity Assay

Journal: Nature

Article Title: Cytoplasmic control of intranuclear polarity by human cytomegalovirus

doi: 10.1038/s41586-020-2714-x

Figure Lengend Snippet: a, Emerin is polarized in HCMV-infected cells. Lines represent mean ± SEM; n= 19,428 cells total from 3 independent biological replicates. b, Onset of Emerin polarization and formation of nuclear F-actin is detectable by 24h, as nuclear rotation begins. Emerin polarity is established over the nuclear rotation period and is sustained, while F-actin dissipates after the rotation phase. c-d, Stills from - showing nuclear F-actin formation during mock or HCMV (TB40/E-UL99-mCherry) infection of NHDFs expressing nuclear actin chromobody (nAC-TagGFP). Early and late stages of infection are shown in c and d, respectively. e , Expression of actin-binding mutants (m151, m175) of Emerin impairs nuclear F-actin formation. Representative images and quantification of nuclear F-actin (nAC) frequency are shown for each condition; bars represent mean ± SEM, n = 1,230 cells total, **p≤0.01, two-tailed student’s t-test. All data shown is representative of 3 independent biological replicates.

Article Snippet: The viral vector encoding a nuclear actin chromobody (actin targeting nanobody tagged with TagGFP2 and a nuclear localization signal; Chromotek: acg-n) was generated by cloning the chromobody sequence into the pWPXL lentiviral vector (Addgene: #12257) using nAC-for (5’-CGCACCGGTGGATCCGCCACCATGGCTCAGGTGCAGCTGGTGG-3’) and nAC-rev (5’-CCGGAATTCACTAGTTTATTACACCTTCCGCTTTTTCTT-3’) primers to generate pWPXL-TagGFP2-nAC, as previously described .

Techniques: Infection, Expressing, Binding Assay, Two Tailed Test

Journal: Nature

Article Title: Cytoplasmic control of intranuclear polarity by human cytomegalovirus

doi: 10.1038/s41586-020-2714-x

Figure Lengend Snippet: a, Emerin is polarized in HCMV-infected cells. Lines represent mean ± SEM; n= 19,428 cells total from 3 independent biological replicates. b, Onset of Emerin polarization and formation of nuclear F-actin is detectable by 24h, as nuclear rotation begins. Emerin polarity is established over the nuclear rotation period and is sustained, while F-actin dissipates after the rotation phase. c-d, Stills from - showing nuclear F-actin formation during mock or HCMV (TB40/E-UL99-mCherry) infection of NHDFs expressing nuclear actin chromobody (nAC-TagGFP). Early and late stages of infection are shown in c and d, respectively. e , Expression of actin-binding mutants (m151, m175) of Emerin impairs nuclear F-actin formation. Representative images and quantification of nuclear F-actin (nAC) frequency are shown for each condition; bars represent mean ± SEM, n = 1,230 cells total, **p≤0.01, two-tailed student’s t-test. All data shown is representative of 3 independent biological replicates.

Article Snippet: Similarly, the viral vector encoding a histone chromobody (Histone H2A-H2B heterodimer targeting nanobody tagged with eGFP; Chromotek: tcg) was generated by cloning the chromobody sequence into pWPXL using the following primers: Histone-Chromobody-for (5’-TATCCTCGAGTACGCGTGCCACCATGGCCCAGGTGCAGCTG-3’) Histone-Chromobody-rev (5’- TTTCCCGGGAATTCTTATTACTTGTACAGCTCGTCCATGCC--3’).

Techniques: Infection, Expressing, Binding Assay, Two Tailed Test

Journal: Technology in Cancer Research & Treatment

Article Title: In Vivo Visualized Tracking of Tumor-Derived Extracellular Vesicles Using CRISPR-Cas9 System

doi: 10.1177/15330338221085370

Figure Lengend Snippet: The design and characterization of EV tracking system. (A) Schematic of EV tracking system. This system consisted of donor A549 cells that expressed sgRNAs and Cas9 proteins and recipient cells that contained STOP-FP elements. (B) Schematic of the recuperation of FP expression in reporter cells. (C) Schematic of engineered EVs. FP Nb and its affinitive Nb were fused with exosomal membrane protein CD63 and Cas9 proteins, respectively. Cas9 proteins were captured by EVs through the bond of FP-FP Nb, selectively being sorted into EVs. (D) qRT-PCR analysis of sgRNA pair levels in EVs derived from wild-type A549 or donor A549 cells (n = 3 in each group). (E) Western blot analysis of Cas9 protein levels in EVs derived from wild-type A549 cells or donor A549 cells (n = 3 in each group). (F) RNA immunoprecipitation (IP) assay of exosomal Cas9 and sgRNAs derived from wild-type A549 cells or donor A549 cells. Cas9 proteins were tagged with Flag. Lysates of EVs were blotted using Flag antibody (input) or immunoprecipitated with Flag and blotted using Flag antibody (IP). IP with anti-IgG served as control. (G) sgRNAs analysis of immunoprecipitants following IP assay in (F). Abbreviations: EV: extracellulaer vesicle; FP: fluorescent protein; Nb: nanobody; sgRNAs: single-guide RNAs.

Article Snippet: The mCherry/GFP nanobody (Nb) sequence was synthesized from Genescript (Nanjing) and cloned into the lentiCRISPRv2 vector. mCherry or green fluorescent protein (GFP) was amplified by PCR using primers for flanking and cloned into the psin-puromycin vector (Addgene) between MluI and SpeI restriction sites; CD63 was amplified by PCR, and the amplicons were cloned into psin-mCherry/GFP vector.

Techniques: Expressing, Membrane, Quantitative RT-PCR, Derivative Assay, Western Blot, RNA Immunoprecipitation, Immunoprecipitation, Control

Journal: Cell Chemical Biology

Article Title: Directed evolution of potent neutralizing nanobodies against SARS-CoV-2 using CDR-swapping mutagenesis

doi: 10.1016/j.chembiol.2021.05.019

Figure Lengend Snippet: Summary of the discovery and affinity maturation of nanobodies against the spike protein of SARS-CoV-2 A synthetic nanobody library displayed on yeast was screened against the receptor-binding domain (RBD), spike (S1) protein, and spike protein trimer of SARS-CoV-2 by MACS and FACS. Two lead clones (KA1 and KC3) were identified and affinity matured using error-prone PCR. The sublibraries were screened against the S1 protein by FACS to isolate nanobody variants (KA1.ep1, KC3.ep3, and KC3.ep5) with superior binding activity relative to a potent neutralizing nanobody generated via immunization (Ty1).

Article Snippet: Nanobody-Fc expression plasmid was digested with NheI-HF and HindIII-HF, as instructed by the manufacturer’s protocol, followed by treatment with calf intestinal alkaline phosphatase (New England Biolabs, M0525L).

Techniques: Binding Assay, Clone Assay, Activity Assay, Generated

Journal: Cell Chemical Biology

Article Title: Directed evolution of potent neutralizing nanobodies against SARS-CoV-2 using CDR-swapping mutagenesis

doi: 10.1016/j.chembiol.2021.05.019

Figure Lengend Snippet: Affinity-matured nanobodies possess a combination of CDRs from the two lead clones (A) Affinity maturation of lead nanobodies KA1 and KC3 via error-prone PCR results in nanobody variants that possess one CDR from each lead nanobody (CDR2 [red] from KA1 and CDR3 [blue] from KC3) in addition to one CDR (CDR1 [green]) that differs by only a single mutation. (B) Nanobody sequences (Kabat numbering) for the three affinity-matured variants. Residues that are different from KA1.ep1 are indicated with an amino acid letter.

Article Snippet: Nanobody-Fc expression plasmid was digested with NheI-HF and HindIII-HF, as instructed by the manufacturer’s protocol, followed by treatment with calf intestinal alkaline phosphatase (New England Biolabs, M0525L).

Techniques: Clone Assay, Mutagenesis

Journal: Cell Chemical Biology

Article Title: Directed evolution of potent neutralizing nanobodies against SARS-CoV-2 using CDR-swapping mutagenesis

doi: 10.1016/j.chembiol.2021.05.019

Figure Lengend Snippet: Affinity-matured nanobodies potently neutralize SARS-CoV-2 pseudovirus and live virus (A) Neutralization results for nanobodies as bivalent Fc-fusion proteins (KA1, KC3, KA1.ep1, KC3.ep3, KC3.ep5, and Ty1) and an antibody (CB6) for inhibiting pseudovirus infectivity in a luciferase-based, HEK293T reporter cell line. Pseudovirus particles were preincubated with antibodies and added to reporter cells, and luciferase signal was measured after 48 h. (B) Neutralization results for nanobodies as bivalent Fc-fusion proteins (KC3.ep3, Ty1) and antibodies (CB6) for inhibiting live virus infection of VeroE6 cells. Nanobody and antibody dilutions were tested in eight replicate wells each. After cells were incubated with virus and nanobodies or antibody for 3 days, the cells were examined microscopically for visible cytopathic effect. Wells with any degree of visible, virus-induced cytopathic effect were scored as positive for infection. In (A), the data are averages of four or five repeats, and the error bars are standard deviations. In (B), the data are averages of two to four repeats, and the error bars are standard deviations.

Article Snippet: Nanobody-Fc expression plasmid was digested with NheI-HF and HindIII-HF, as instructed by the manufacturer’s protocol, followed by treatment with calf intestinal alkaline phosphatase (New England Biolabs, M0525L).

Techniques: Neutralization, Infection, Luciferase, Incubation

Journal: Cell Chemical Biology

Article Title: Directed evolution of potent neutralizing nanobodies against SARS-CoV-2 using CDR-swapping mutagenesis

doi: 10.1016/j.chembiol.2021.05.019

Figure Lengend Snippet: Affinity-matured nanobody recognizes an epitope in the receptor-binding domain that overlaps with epitopes recognized by ACE2 and other potent SARS-CoV-2 neutralizing nanobodies and antibodies Bivalent nanobodies (KC3.ep3, VHH-72, and Ty1), antibodies (S309, CR3022, CB6, and C119) and ACE2 were preincubated with biotinylated receptor-binding domain of SARS-CoV-2 (5 nM) over a range of nanobody, antibody, and ACE2 concentrations, and then the mixtures were added to yeast cells displaying monovalent KC3.ep3. The percentage bound receptor-binding domain is reported relative to the amount bound in the absence of preblocking. The results are averages from three independent repeats, and the error bars are standard deviations.

Article Snippet: Nanobody-Fc expression plasmid was digested with NheI-HF and HindIII-HF, as instructed by the manufacturer’s protocol, followed by treatment with calf intestinal alkaline phosphatase (New England Biolabs, M0525L).

Techniques: Binding Assay

Journal: Cell Chemical Biology

Article Title: Directed evolution of potent neutralizing nanobodies against SARS-CoV-2 using CDR-swapping mutagenesis

doi: 10.1016/j.chembiol.2021.05.019

Figure Lengend Snippet:

Article Snippet: Nanobody-Fc expression plasmid was digested with NheI-HF and HindIII-HF, as instructed by the manufacturer’s protocol, followed by treatment with calf intestinal alkaline phosphatase (New England Biolabs, M0525L).

Techniques: Recombinant, Luciferase, Protease Inhibitor, DC Protein Assay, Plasmid Preparation, Software, Magnetic Beads, Gel Extraction, Purification

Journal: The Journal of Biological Chemistry

Article Title: Nanobodies targeting EGFR provide insight into conformations stabilized by glioblastoma mutations

doi: 10.1016/j.jbc.2025.110374

Figure Lengend Snippet: Soluble EGFR used as antigen in yeast-displayed nanobody selections . A , dimerization of cell-surface EGFR by ligand is allosterically transmitted to the kinase domains, which form an asymmetric dimer, with one active monomer that can trans-phosphorylate tyrosines in the C-terminal tail. B , the sEGFR Fc-fusion monomer (sEGFR-Fc) and ∗ C ) the Fc-fusion homodimer ((sEGFR) 2 -Fc) were cleaved with IdeS protease, yielding the EGFR ectodomain molecularly fused to the Fc hinge. D , gel filtration before and after IdeS cleavage. left : Gel filtration on an S200 column shows the (sEGFR) 2 -Fc elutes as a single monodisperse peak before and after ideS cleavage. Right : The sEGFR-Fc was generated by co-transfecting plasmids containing EGFR Fc-fusion and Fc. This yielded three separate species (sEGFR) 2 -Fc, sEGFR-Fc, and Fc , which were separated by gel filtration using two S200 columns in series. E , isothermal titration calorimetry (ITC) experiments demonstrate (sEGFR) 2 -Fc binds EGF with a Kd of 12 ± 1.1 nM, has N value of 0.73 ± 0.0016 Sites, ΔH of 23,000 ± 89 cal/mol, and ΔS of 115 cal/mol/deg.

Article Snippet: After gel purification, the resulting PCR products served as templates for two final PCRs, performed in parallel for IgG2 and IgG3 nanobody genes. pCTcon2 vector (Addgene, Plasmid # 41843) was cleaved with NheI and BamHI endonucleases (New England Biolabs).

Techniques: Filtration, Generated, Isothermal Titration Calorimetry

Journal: The Journal of Biological Chemistry

Article Title: Nanobodies targeting EGFR provide insight into conformations stabilized by glioblastoma mutations

doi: 10.1016/j.jbc.2025.110374

Figure Lengend Snippet: Enrichment of yeast libraries for nanobodies binding to (sEGFR) 2 -hinge . A , yeast cells expressing a library of nanobodies were incubated with positive and negative selection antigens. Yeast cells expressing nanobodies binding to positive selection antigen were kept for future rounds of sorting while those not binding to antigen or binding to negative selection antigen were removed from the library. B , yeast displayed nanobody libraries were depleted for binders to sEGFR-hinge and enriched for binders to (sEGFR) 2 -hinge in the absence ( left ) or presence ( right ) of EGF ligand through one MACS and three rounds of FACS. Decreasing concentrations of ligand were used to enrich for high affinity binders. This sorting strategy is representative of two of three selections in which kinetic selection was done .

Article Snippet: After gel purification, the resulting PCR products served as templates for two final PCRs, performed in parallel for IgG2 and IgG3 nanobody genes. pCTcon2 vector (Addgene, Plasmid # 41843) was cleaved with NheI and BamHI endonucleases (New England Biolabs).

Techniques: Binding Assay, Expressing, Incubation, Selection

Journal: The Journal of Biological Chemistry

Article Title: Nanobodies targeting EGFR provide insight into conformations stabilized by glioblastoma mutations

doi: 10.1016/j.jbc.2025.110374

Figure Lengend Snippet: Nanobody selectivity elucidates distinct conformational states stabilized by GBM mutations . A , single concentration measurements of antibodies binding to oncogenic EGFR mutants on the cell surface show distinct binding characteristics. Measurements were performed in triplicate at WT EC 50 concentrations. Values are normalized to expression (LA22 binding) and to WT binding. Asterisks indicate statistically significant binding compared to WT, determined using unpaired t-tests with Bonferroni correction, p -value: ∗, < 0.0125; ∗∗, < 0.00125. Error bars show S.D. B , cell-surface antibody titrations on oncogenic mutants further demonstrate the distinct characteristics shown in A . Values are normalized to expression (LA22 binding). All measurements were performed on transiently transfected Expi293 F cells expressing either WT EGFR or GBM mutations.

Article Snippet: After gel purification, the resulting PCR products served as templates for two final PCRs, performed in parallel for IgG2 and IgG3 nanobody genes. pCTcon2 vector (Addgene, Plasmid # 41843) was cleaved with NheI and BamHI endonucleases (New England Biolabs).

Techniques: Concentration Assay, Binding Assay, Expressing, Transfection

Journal: The Journal of Biological Chemistry

Article Title: Nanobodies targeting EGFR provide insight into conformations stabilized by glioblastoma mutations

doi: 10.1016/j.jbc.2025.110374

Figure Lengend Snippet: Summary of HD nanobody binding characteristics . A , binding preferences. B , epitopes. Groups 1 and 5 bind vIII and therefore their epitopes must reside in its segment of EGFR. As with other group 3 nanobodies, the HD25 epitope likely includes the ligand binding site of domain III; HD25 is strongly competitive with EGF and 7D12, which each bind domain III. As with the structurally characterized group 4 nanobody EgB4, HD30 likely binds domains I or II. HD30 does not bind vIII and is strongly competitive with EgB4 which binds across domain I and II.

Article Snippet: After gel purification, the resulting PCR products served as templates for two final PCRs, performed in parallel for IgG2 and IgG3 nanobody genes. pCTcon2 vector (Addgene, Plasmid # 41843) was cleaved with NheI and BamHI endonucleases (New England Biolabs).

Techniques: Binding Assay, Ligand Binding Assay