Journal: Respiratory Research

Article Title: Nasal lavage natural killer cell function is suppressed in smokers after live attenuated influenza virus

doi: 10.1186/1465-9921-12-102

Figure Lengend Snippet: Antibodies Used

Article Snippet: Anti-human CD56 [ ] , Mouse IgG2b, κ , 1:100 , RnD Systems , 301021 , MAB24081.

Techniques: Plasmid Preparation

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Sustained Endocytosis Inhibition via Locally-Injected Drug-Eluting Hydrogel Improves ADCC-Mediated Antibody Therapy in Colorectal Cancer.

doi: 10.1002/advs.202407239

Figure Lengend Snippet: Figure 6. Anti-tumor activity of Gel@Cmab/PCZ in an EGFR-internalized CRC PDX tumor model. A, B) Schematic illustration of the experimental design used to evaluate antitumor activity in vivo using partially humanized CRC-PDX mice (A), which were derived from a sample of patient with primary CRC featuring EGFR internalization and the treatment schedule (B). C) Representative tumor images on day 25 (n = 6). D, E) Average and individual tumor growth curves of CRC-PDX after various treatments (n = 6). F) The weight of excised CRC-PDX after various treatments (n = 6). G) Representative H&E, Ki67, p-Erk1/2, and p-Akt staining images of tumor tissues after different treatments. Scale bars, 50 μm. H) Representative CD56 staining images of tumor tissues after different treatments. Scale bars, 50 μm. I) The percentage of NK cells (CD56+) in isolated tumor tissues after treatment (n = 6). J, K) Representative flow cytometry plots (J) and quantification (K) of the population of CD69+ activated NK cells in isolated tumor tissues after treatment (n = 6). Data are presented as mean ± S.D. Statistical significance was calculated using two-way ANOVA (D) or the one-way ANOVA (F, I, K). ns, non-significant, **p < 0.01, ****p < 0.0001.

Article Snippet: Materials: Invitrogen (USA) provided Epidermal growth factor labeled with biotin, bound to Alexa FluorTM 488 streptavidin (known as Alexa FluorTM 488 EGF complex, #E13345), Goat anti-Human IgG (H+L) Cross-Adsorbed Secondary Antibody (Alexa FluorTM 555, #A21433), Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody (Alexa FluorTM 555, #A21429), ProLongTM Gold antifade reagent (#P10144), and Penicillin-Streptomycin (#15 070 063).Antibodies against EGFR (#4267), Phospho-EGFR (Tyr1068) (#3777), Ki-67 (#9449), cleaved caspase-3 (#9661s), CD56 (#99 746), Akt (#4691), p44/42 MAPK (Erk1/2) (#4695), Phospho-Akt (Ser473) (#4060), Phospho-p44/42 MAPK (Erk1/2) (Thr202/.Tyr204 (#4370), pancytokeratin (#4545S), and GAPDH (#2118) were acquired from Cell Signaling Technology (CST) in the United States.

Techniques: Activity Assay, In Vivo, Derivative Assay, Staining, Isolation, Cytometry

Journal: Cell death & disease

Article Title: Scutellarin activates IDH1 to exert antitumor effects in hepatocellular carcinoma progression.

doi: 10.1038/s41419-024-06625-6

Figure Lengend Snippet: Fig. 7 Scu exerts antitumor effects by promoting IDH1 enzyme activity and activating the tumor immune microenvironment in vivo. A Schematic plan for the administration of Scu (60 and 100 mg/kg/day). C The tumor weight and D tumor volume were monitored every week for four weeks. After the mice were sacrificed, the resected tumors were B photographed and processed for pathological and immunohistochemical assays for E, F necrosis area and Ki67, L, M HIF1α, GLUT1, VEGFA, and N, O CD4, CD8, F4/80, CD56 and PD-L1 expression. G The relative mRNA levels of the indicated genes, H, I the expression levels of the indicated proteins, J the level of α-KG, and K IDH1 activity in tumor tissue were detected after treatment with Scu in vivo. The black arrows indicate positively stained cells. Scale bars, 50 μm. n ≥5. The data are mean ± SD; *P < 0.05; **P < 0.01; ***P < 0.001 compared to the control group.

Article Snippet: The sections were blocked in PBS containing 10% normal goat serum and 0.3% Triton X-100 for 60min; labeled with primary antibodies against Ki67 (1:2000, 27309-1-AP, Proteintech), IDH1 (1:200, 12332-1-AP, Proteintech), HIF1a (1:200, A22041, ABclonal), GLUT1 (1:200, 81463-1-RR, Proteintech), VEGFA (1:100, A12303, ABclonal), CD4 (1:500, 67786-1-Ig, Proteintech), CD8 (1:10,000, 66868-1-Ig, Proteintech), F4/80 (1:100, A23788, ABclonal), CD56 (1:2000, 14255-1-AP, Proteintech), and PDL1 (1:1000, 28076-1-AP, Proteintech) overnight at 4 °C; and incubated with the corresponding goat secondary antibodies for 1 h at room temperature.

Techniques: Activity Assay, In Vivo, Immunohistochemical staining, Expressing, Staining, Control

Journal: Cell Reports Medicine

Article Title: Autoantibodies against NCAM1 from patients with schizophrenia cause schizophrenia-related behavior and changes in synapses in mice

doi: 10.1016/j.xcrm.2022.100597

Figure Lengend Snippet: Identification of anti-NCAM1 autoantibodies (A) Titers of anti-NCAM1 autoantibodies in serum by ELISA. ∗∗p < 0.01 (n = 201, healthy controls; n = 223, patients with schizophrenia; Mann-Whitney U test). (B) Immunocytochemistry using a commercial anti-NCAM1 antibody, serum and CSF from schizophrenia patient 1, and serum from healthy controls. NCAM1 and EGFP were expressed from a plasmid. Confocal images show antibodies bound to the membrane of EGFP-positive HeLa cells. Similar results were obtained for all anti-NCAM1 antibody-positive patients with schizophrenia ( Figure S1 B). Antibodies in serum did not react with EGFP because (1) they did not react with EGFP in the nucleus and (2) they did not react with cells transfected with an empty plasmid expressing only EGFP (data not shown). Scale bar: 10 μm. Ab, antibody; Sz, schizophrenia. (C) Titers of anti-NCAM1 autoantibodies in serum by cell-based assay. ∗∗p < 0.01 (n = 201, healthy controls; n = 223, patients with schizophrenia; Mann-Whitney U test). (D) ELISA analysis of serum-soluble NCAM in healthy controls (n = 201) and patients with schizophrenia (n = 223). ∗∗p < 0.01 (Mann-Whitney U test). (E) ELISA analysis of serum-soluble NCAM in schizophrenia patients with (n = 211) or without (n = 12) anti-NCAM1 autoantibodies defined by cell-based assay. ∗p < 0.05 (Mann-Whitney U test). (F) ELISA analysis of serum-soluble NCAM in healthy controls (n = 201) and schizophrenia patients without anti-NCAM1 autoantibodies (n = 211) defined by cell-based assay. ∗∗p < 0.01 (Mann-Whitney U test).

Article Snippet: Polystyrene microtiter plates (3455, Thermo Scientific, Waltham, MA, USA) were coated with 100 μL (2 μg/mL) of NCAM1 recombinant protein (10673-H08H, Sino Biological) in TBS buffer and incubated overnight at 4°C.

Techniques: Enzyme-linked Immunosorbent Assay, MANN-WHITNEY, Immunocytochemistry, Plasmid Preparation, Membrane, Transfection, Expressing, Cell Based Assay

Journal: Cell Reports Medicine

Article Title: Autoantibodies against NCAM1 from patients with schizophrenia cause schizophrenia-related behavior and changes in synapses in mice

doi: 10.1016/j.xcrm.2022.100597

Figure Lengend Snippet: The main epitope recognized by anti-NCAM1 antibodies in schizophrenia resides within the Ig1 domain (A) NCAM1 deletion constructs. (B) Immunocytochemistry using serum from patient 1 with schizophrenia, who was positive for anti-NCAM1 autoantibodies. NCAM1 deletion constructs and EGFP were expressed from a plasmid. Similar results were obtained from all anti-NCAM1 antibody-positive patients with schizophrenia. Scale bar: 10 μm. (C) Immunocytochemical confirmation of the expression of NCAM1ΔIg1 and NCAM1ΔIg1–5 using a commercial anti-NCAM1 antibody. Scale bar: 10 μm. (D) Western blot analysis of deletion constructs of NCAM1 transfected into HeLa cells revealed that the main epitope recognized by anti-NCAM1 autoantibodies is in the Ig1 domain.

Article Snippet: Polystyrene microtiter plates (3455, Thermo Scientific, Waltham, MA, USA) were coated with 100 μL (2 μg/mL) of NCAM1 recombinant protein (10673-H08H, Sino Biological) in TBS buffer and incubated overnight at 4°C.

Techniques: Construct, Immunocytochemistry, Plasmid Preparation, Expressing, Western Blot, Transfection

Journal: Cell Reports Medicine

Article Title: Autoantibodies against NCAM1 from patients with schizophrenia cause schizophrenia-related behavior and changes in synapses in mice

doi: 10.1016/j.xcrm.2022.100597

Figure Lengend Snippet: Anti-NCAM1 autoantibodies disrupt NCAM1-NCAM1 and NCAM1-GDNF interactions (A) Pull-down assay confirming that IgG purified from a patient with schizophrenia who was positive for anti-NCAM1 autoantibodies disrupts NCAM1-NCAM1 interactions. His-tagged proteins were pulled down by Ni-NTA-agarose, and GST-tagged proteins were pulled down by Glutathione Sepharose. (B) Pull-down assay showing that IgG purified from a patient with schizophrenia who was anti-NCAM1 autoantibody-positive disrupts the NCAM1-GDNF interaction. His-tagged proteins were pulled down by Ni-NTA-agarose, and GST-tagged proteins were pulled down by Glutathione Sepharose.

Article Snippet: Polystyrene microtiter plates (3455, Thermo Scientific, Waltham, MA, USA) were coated with 100 μL (2 μg/mL) of NCAM1 recombinant protein (10673-H08H, Sino Biological) in TBS buffer and incubated overnight at 4°C.

Techniques: Pull Down Assay, Purification

Journal: Cell Reports Medicine

Article Title: Autoantibodies against NCAM1 from patients with schizophrenia cause schizophrenia-related behavior and changes in synapses in mice

doi: 10.1016/j.xcrm.2022.100597

Figure Lengend Snippet: Injection of anti-NCAM1 autoantibodies from a patient with schizophrenia into mice (A) Experimental protocol for IgG injection. AAV1-SYN1-EGFP and AAV2-VAMP2-mCherry were injected into the frontal cortex of mice aged 6 weeks, and purified IgG was injected into the CSF of mice aged 8 weeks. Molecular, histological, two-photon microscopy, and behavioral analyses were performed in 9-week-old mice. IHC, immunohistochemistry; IP, immunoprecipitation; WB, western blot. (B) Immunoprecipitation analysis of tissue from the frontal cortex of mice revealed that the NCAM1-Fyn interaction was inhibited by anti-NCAM1 autoantibodies acquired from patients with schizophrenia. CT, computed tomography. (C) Effect of IgG purified from patient 1 with schizophrenia on FAK, MEK1, and ERK1 phosphorylation in the frontal cortex. Removal of anti-NCAM1 antibodies from purified IgG reversed the decrease in pFAK, pMEK, and pERK1. (D) Quantitative analyses of western blots with five mice per group. ∗∗p < 0.01 (n = 5, Tukey’s honest significant difference [HSD] test). Data are expressed as the mean ± SEM. (E) Two-photon microscopic images of dendritic spines in the first layer of the frontal cortex of mice injected with AAV1-SYN1-EGFP and IgG purified from patient 1 with schizophrenia or IgG purified from a healthy control. Removal of anti-NCAM1 antibodies from the purified IgG reversed the decrease in the number of spines. The graph on the right shows quantitative analysis of spine number. ∗∗p < 0.01 (n = 5 mice per group; 50 dendrites/mouse, 500 spines/mouse; Tukey’s HSD test). Data are expressed as the mean ± SEM. Scale bar: 5 μm. (F) Two-photon microscopic images showing contact between axon terminals and dendritic spines in the first layer of the frontal cortex of mice injected with AAV2-VAMP2-mCherry, AAV1-SYN1-EGFP, IgG purified from patient 1 with schizophrenia, or IgG from a healthy control. The graph on the right shows quantitative analysis of axon terminals merged with spines. ∗∗p < 0.01 (n = 5 mice per group; 50 dendrites/mouse, 500 spines/mouse; Tukey’s HSD test). Data are expressed as the mean ± SEM. Scale bar: 5 μm. (G) Alteration ratios in the Y maze test after injection of purified IgG from patient 1 with schizophrenia or from a healthy control. Removal of anti-NCAM1 antibodies from purified IgG reversed the decrease in the alteration ratios. ∗∗p < 0.01 (n = 9 mice per group; Tukey’s HSD test). Data are expressed as the mean ± SEM. (H) Pre-pulse inhibition rates of mice injected with IgG purified from patient 1 with schizophrenia or a healthy control. Removal of anti-NCAM1 antibodies from purified IgG reversed the deficiency in pre-pulse inhibition. ∗p < 0.05 and ∗∗p < 0.01 (n = 9 mice per group; Tukey’s HSD test). Data are expressed as the mean ± SEM.

Article Snippet: Polystyrene microtiter plates (3455, Thermo Scientific, Waltham, MA, USA) were coated with 100 μL (2 μg/mL) of NCAM1 recombinant protein (10673-H08H, Sino Biological) in TBS buffer and incubated overnight at 4°C.

Techniques: Injection, Purification, Microscopy, Immunohistochemistry, Immunoprecipitation, Western Blot, Computed Tomography, Control, Inhibition

Journal: Cell Reports Medicine

Article Title: Autoantibodies against NCAM1 from patients with schizophrenia cause schizophrenia-related behavior and changes in synapses in mice

doi: 10.1016/j.xcrm.2022.100597

Figure Lengend Snippet: Anti-NCAM1 autoantibodies from patients with schizophrenia cause schizophrenia-related behavior and changes in synapse numbers in mice (A) Experimental protocol for IgG injection. AAV1-SYN1-EGFP and AAV2-VAMP2-mCherry were injected into the frontal cortex of mice aged 6 weeks, and purified IgG was injected into the CSF of mice aged 8 weeks. Two-photon microscopy and behavioral analyses were performed in 9-week-old mice. (B) Two-photon microscopic analysis of dendritic spines in the first layer of the frontal cortex of mice injected with AAV1-SYN1-EGFP and IgG purified from a healthy control and patient 2 and patient 3 with schizophrenia. ∗∗p < 0.01 (n = 5 mice per group; 50 dendrites/mouse, 500 spines/mouse; Tukey’s HSD test). Data are expressed as the mean ± SEM. (C) Two-photon microscopic analysis of axon terminals merged with spines in the first layer of the frontal cortex of mice injected with AAV2-VAMP2-mCherry, AAV1-SYN1-EGFP, and IgG purified from a healthy control and patient 2 and patient 3 with schizophrenia. ∗∗p < 0.01 (n = 5 mice per group; 50 dendrites/mouse, 500 spines/mouse; Tukey’s HSD test). Data are expressed as the mean ± SEM. Scale bar: 5 μm. (D) Alteration ratios in the Y maze test after injection of IgG purified from a healthy control and patient 2 and patient 3 with schizophrenia. ∗∗p < 0.01 (n = 9 mice per group; Tukey’s HSD test). Data are expressed as the mean ± SEM. (E) Pre-pulse inhibition rates of mice injected with IgG purified from a healthy control and patient 2 and patient 3 with schizophrenia. ∗p < 0.05 and ∗∗p < 0.01 (n = 9 mice per group; Tukey’s HSD test). Data are expressed as the mean ± SEM.

Article Snippet: Polystyrene microtiter plates (3455, Thermo Scientific, Waltham, MA, USA) were coated with 100 μL (2 μg/mL) of NCAM1 recombinant protein (10673-H08H, Sino Biological) in TBS buffer and incubated overnight at 4°C.

Techniques: Injection, Purification, Microscopy, Control, Inhibition

Journal: Cell Reports Medicine

Article Title: Autoantibodies against NCAM1 from patients with schizophrenia cause schizophrenia-related behavior and changes in synapses in mice

doi: 10.1016/j.xcrm.2022.100597

Figure Lengend Snippet:

Article Snippet: Polystyrene microtiter plates (3455, Thermo Scientific, Waltham, MA, USA) were coated with 100 μL (2 μg/mL) of NCAM1 recombinant protein (10673-H08H, Sino Biological) in TBS buffer and incubated overnight at 4°C.

Techniques: Virus, Plasmid Preparation, Recombinant, Labeling, Enzyme-linked Immunosorbent Assay, Software