Journal: International Journal of Molecular Medicine

Article Title: Overexpression of FcγRIIB regulates downstream protein phosphorylation and suppresses B cell activation to ameliorate systemic lupus erythematosus

doi: 10.3892/ijmm.2020.4698

Figure Lengend Snippet: (A) Observation of double-resistant HT-1080 cells by fluorescence microscopy (magnification ×400). The expression of human mFcγRIIB in HT-1080 cells was detected by immunofluorescence staining. Light, light field; green, green fluorescent protein (GFP) expression in cells; red, mFcγRIIB expression in cells; merge, GFP and mFcγRIIB expression in cells. The experiments were repeated 3 times. (B) The protein expression levels of sFcγRIIB in human were examined by western blot analysis, with β-actin as a loading control. Lane 1, 0 ng/ml Dox inducer; lane 2, 100 ng/ml Dox inducer; lane 3, 500 ng/ml Dox inducer; lane 4, 1,000 ng/ml Dox inducer. * P<0.05. Data are presented as the means ± SD of 3 independent experiments. Dox, doxycycline; mFcγRIIB, membrane-bound type FcγRIIB; sFcγRIIB, soluble FcγRIIB.

Article Snippet: Serum FcγRIIB-IgG was assayed using rabbit anti-mouse FcγRIIB monoclonal antibody (cat. no. sc-365864; 1:2,000; Santa Cruz Biotechnology, Inc.) and HRP-labeled goat anti-mouse IgG antibody (cat. no. bs-0296G-HRP; 1:5,000; Beijing Bioss Biotechnology Co., Ltd.).

Techniques: Fluorescence, Microscopy, Expressing, Immunofluorescence, Staining, Western Blot, Control, Membrane

Journal: International Journal of Molecular Medicine

Article Title: Overexpression of FcγRIIB regulates downstream protein phosphorylation and suppresses B cell activation to ameliorate systemic lupus erythematosus

doi: 10.3892/ijmm.2020.4698

Figure Lengend Snippet: (A) Fluorescence microscopy to observe the expression of mFcγRIIB in B lymphocytes (magnification 400x). B cells were transfected using mFcγRIIB-lentivirus for 72 h. The infection efficiency of mFcγRIIB-lentivirus was observed by fluorescence microscopy. No-Lentv-SLE, no transfection in the SLE patients; Lentv-SLE, mFcγRIIB-lentivirus transfection in the SLE patients; No-Lentv-health, no transfection in the healthy controls; Lentiv-health, mFcγRIIB-lentivirus transfection in the healthy controls. Light, light field; green, green fluorescent protein (GFP) expression in cells; red, mFcγRIIB expres-sion in cells; merge, GFP and mFcγRIIB expression in cells. The experiments were repeated 3 times. (B) The protein expression levels of mFcγRIIB in human were examined by western blot analysis, with β-actin as a loading control. Lane 1, Control SLE group; lane 2, virus-infected SLE group; lane 3, healthy control group; lane 4, virus-infected healthy group. * P<0.05. Data are presented as the means ± SD of 3 independent experiments. mFcγRIIB, membrane-bound type FcγRIIB; sFcγRIIB, soluble FcγRIIB.

Article Snippet: Serum FcγRIIB-IgG was assayed using rabbit anti-mouse FcγRIIB monoclonal antibody (cat. no. sc-365864; 1:2,000; Santa Cruz Biotechnology, Inc.) and HRP-labeled goat anti-mouse IgG antibody (cat. no. bs-0296G-HRP; 1:5,000; Beijing Bioss Biotechnology Co., Ltd.).

Techniques: Fluorescence, Microscopy, Expressing, Transfection, Infection, Western Blot, Control, Virus, Membrane

Journal: International Journal of Molecular Medicine

Article Title: Overexpression of FcγRIIB regulates downstream protein phosphorylation and suppresses B cell activation to ameliorate systemic lupus erythematosus

doi: 10.3892/ijmm.2020.4698

Figure Lengend Snippet: (A) ELISA detection of human IgG antibody levels. No-Lentv-SLE, control SLE grou; Lentv-SLE, virus-infected SLE group; No-Lentv-Health, healthy control group; Lentv-Health, virus-infected healthy group; CtDNA, calf thymus DNA. IC, anti-calf thymus DNA-immune complexes. * P<0.05; ** P<0.01. The experiments were repeated 3 times. (B and C) The human protein expression levels of BTK, p-BTK, DOK1, p-DOK1, Lyn, p-Lyn, SHIP and p-SHIP were examined by western blot analysis, with β-actin as a loading control. The phosphorylation and total protein levels of BTK, DOK1, Lyn and SHIP were detected in B cells from patients with SLE and healthy subjects infected with mFcγRIIB lentivirus following anti-ctDNA-IC stimulation, and the relative level for each phosphorylation-protein/each respective total protein was calculated. No-Lentv-SLE, control SLE group; Lentv-SLE, virus-infected SLE group; No-Lentv-Health, healthy control group; Lentv-Health, virus-infected healthy group. * P<0.05; ** P<0.01. Data are presented as the means ± SD of 3 independent experiments. SLE, systemic lupus erythematosus; mFcγRIIB, membrane-bound type FcγRIIB; sFcγRIIB, soluble FcγRIIB; BTK, Bruton's tyrosine kinase; Lyn, Lyn proto-oncogene, Src family tyrosine kinase; DOK-1, docking protein 1; SHIP, inositol polyphosphate-5-phosphatase D.

Article Snippet: Serum FcγRIIB-IgG was assayed using rabbit anti-mouse FcγRIIB monoclonal antibody (cat. no. sc-365864; 1:2,000; Santa Cruz Biotechnology, Inc.) and HRP-labeled goat anti-mouse IgG antibody (cat. no. bs-0296G-HRP; 1:5,000; Beijing Bioss Biotechnology Co., Ltd.).

Techniques: Enzyme-linked Immunosorbent Assay, Control, Virus, Infection, Expressing, Western Blot, Phospho-proteomics, Membrane

Journal: International Journal of Molecular Medicine

Article Title: Overexpression of FcγRIIB regulates downstream protein phosphorylation and suppresses B cell activation to ameliorate systemic lupus erythematosus

doi: 10.3892/ijmm.2020.4698

Figure Lengend Snippet: (A) Apoptotic rate of B cells infected by human mFcγRIIB lentivirus. No-Lentv-SLE, control SLE group; Lentv-SLE, virus-infected SLE group; No-Lentv-Health, healthy control group; Lentv-Health, virus-infected healthy group; CtDNA, calf thymus DNA; IC, anti-calf thymus DNA-immune complexes. * P<0.05; ** P<0.01. The experiments were repeated 3 times. (B and C) The human protein expression levels of BTK, p-BTK, DOK1, p-DOK-1, Lyn, p-Lyn, SHIP and p-SHIP were examined by western blot analysis, with β-actin as a loading control. The phosphorylation and total protein levels of BTK, DOK-1, Lyn and SHIP were detected in B cells of patients with SLE and healthy subjects following treatment with human sFcγRIIB or sFcγRIIB plus ctDNA, and the relative level for each phosphorylation-protein/each respective total protein was calculated. Cell-Control-SLE, B cells of SLE patients; Protein-SLE, B cells of SLE patients treated with sFcγRIIB; Protein + CtDNA-SLE, B cells of SLE patients treated with sFcγRIIB and ctDNA; Cell-Control-Health, B cells of healthy subjects; Protein-Health, B cells of healthy persons treated with sFcγRIIB; Protein + CtDNA-Health, B cells of healthy persons treated with sFcγRIIB and ctDNA. * P<0.05; ** P<0.01. Data are presented as the means ± SD of 3 independent experiments. SLE, systemic lupus erythematosus; mFcγRIIB, membrane-bound type FcγRIIB; sFcγRIIB, soluble FcγRIIB; BTK, Bruton's tyrosine kinase; Lyn, Lyn proto-oncogene, Src family tyrosine kinase; DOK-1, docking protein 1; SHIP, inositol polyphosphate-5-phosphatase D.

Article Snippet: Serum FcγRIIB-IgG was assayed using rabbit anti-mouse FcγRIIB monoclonal antibody (cat. no. sc-365864; 1:2,000; Santa Cruz Biotechnology, Inc.) and HRP-labeled goat anti-mouse IgG antibody (cat. no. bs-0296G-HRP; 1:5,000; Beijing Bioss Biotechnology Co., Ltd.).

Techniques: Infection, Control, Virus, Expressing, Western Blot, Phospho-proteomics, Membrane

Journal: International Journal of Molecular Medicine

Article Title: Overexpression of FcγRIIB regulates downstream protein phosphorylation and suppresses B cell activation to ameliorate systemic lupus erythematosus

doi: 10.3892/ijmm.2020.4698

Figure Lengend Snippet: Soluble FcγRIIB binding to immune complexes, with OD values (n=30, means ± SD).

Article Snippet: Serum FcγRIIB-IgG was assayed using rabbit anti-mouse FcγRIIB monoclonal antibody (cat. no. sc-365864; 1:2,000; Santa Cruz Biotechnology, Inc.) and HRP-labeled goat anti-mouse IgG antibody (cat. no. bs-0296G-HRP; 1:5,000; Beijing Bioss Biotechnology Co., Ltd.).

Techniques: Binding Assay

Journal: International Journal of Molecular Medicine

Article Title: Overexpression of FcγRIIB regulates downstream protein phosphorylation and suppresses B cell activation to ameliorate systemic lupus erythematosus

doi: 10.3892/ijmm.2020.4698

Figure Lengend Snippet: Target detection of MRL/lpr SLE mice following mFcγRIIB treatment. (A) Changes in urine protein levels. (B) Changes in serum anti-dsDNA antibody levels. (C) Changes in serum anti-nuclear antibody levels. (D) Changes in mFcγRIIB levels in B cells. pre-control, Control subgroup in the prevention group; pre-empty virus, empty virus subgroup in the prevention group; pre-100 µ l, 100 µ l mFcγRIIB lentivirus subgroup in the prevention group; pre-200 µ l, 200 µ l mFcγRIIB lentivirus subgroup in the prevention group; tre-control, control subgroup in the treatment group; tre-empty virus, empty virus subgroup in the treatment group; tre-100 µ l, 100 µ l mFcγRIIB lentivirus subgroup in the treatment group; tre-200 µ l, 200 µ l mFcγRIIB lentivirus subgroup in the treatment group. * P<0.05; ** P<0.01; n=10 in each group. SLE, systemic lupus erythematosus; mFcγRIIB, membrane-bound type FcγRIIB; sFcγRIIB, soluble FcγRIIB.

Article Snippet: Serum FcγRIIB-IgG was assayed using rabbit anti-mouse FcγRIIB monoclonal antibody (cat. no. sc-365864; 1:2,000; Santa Cruz Biotechnology, Inc.) and HRP-labeled goat anti-mouse IgG antibody (cat. no. bs-0296G-HRP; 1:5,000; Beijing Bioss Biotechnology Co., Ltd.).

Techniques: Control, Virus, Membrane

Journal: International Journal of Molecular Medicine

Article Title: Overexpression of FcγRIIB regulates downstream protein phosphorylation and suppresses B cell activation to ameliorate systemic lupus erythematosus

doi: 10.3892/ijmm.2020.4698

Figure Lengend Snippet: (A) The protein expression levels of BTK, p-BTK, Lyn, p-Lyn, SHIP and p-SHIP in MRL/lpr SLE mouse B cells were examined by western blot analysis, with β-actin as a loading control. (B and C) The phosphorylation and total protein levels of BTK, Lyn and SHIP were detected in B cells from MRL/lpr SLE mice after the infection of mFcγRIIB lentivirus, and the relative level for each phosphorylation-protein/each respective total protein was calculated. Lane 1: pre-empty virus, empty virus subgroup in the prevention group; lane 2: pre-control, control subgroup in the prevention group; lane 3: pre-100 µ l, 100 µ l mFcγRIIB lentivirus subgroup in the prevention group; lane 4: pre-200 µ l, 200 µ l mFcγRIIB lentivirus subgroup in the prevention group; lane 5: tre-control, control subgroup in the treatment group; lane 6: tre-empty virus, empty virus subgroup in the treatment group; lane 7: tre-100 µ l, 100 µ l mFcγRIIB lentivirus subgroup in the treatment group; lane 8: tre-200 µ l, 200 µ l mFcγRIIB lentivirus subgroup in the treatment group. * P<0.05; ** P<0.01. Data are presented as means ± SD of 3 independent experiments. SLE, systemic lupus erythematosus; mFcγRIIB, membrane-bound type FcγRIIB; sFcγRIIB, soluble FcγRIIB; BTK, Bruton's tyrosine kinase; Lyn, Lyn proto-oncogene, Src family tyrosine kinase; DOK-1, docking protein 1; SHIP, inositol polyphosphate-5-phosphatase D.

Article Snippet: Serum FcγRIIB-IgG was assayed using rabbit anti-mouse FcγRIIB monoclonal antibody (cat. no. sc-365864; 1:2,000; Santa Cruz Biotechnology, Inc.) and HRP-labeled goat anti-mouse IgG antibody (cat. no. bs-0296G-HRP; 1:5,000; Beijing Bioss Biotechnology Co., Ltd.).

Techniques: Expressing, Western Blot, Control, Phospho-proteomics, Infection, Virus, Membrane

Journal: International Journal of Molecular Medicine

Article Title: Overexpression of FcγRIIB regulates downstream protein phosphorylation and suppresses B cell activation to ameliorate systemic lupus erythematosus

doi: 10.3892/ijmm.2020.4698

Figure Lengend Snippet: Target detection in MRL/lpr SLE mice. (A) Detection of sFcγRIIB. (B) Detection of FcγRIIB binding with IgG. (C) Detection of serum anti-double-stranded DNA antibody. (D) Detection of urine protein. pre-control, control subgroup in the prevention group; pre-60 µ l, 4.8 µ g sFcγRIIB subgroup in the prevention group; pre-120 µ l, 9.6 µ g sFcγRIIB subgroup in the prevention group; pre-180 µ l, 14.4 µ g sFcγRIIB subgroup in prevention group; tre-control, control subgroup in the treatment group; tre-60 µ l, 4.8 µ g sFcγRIIB subgroup in the treatment group; tre-120 µ l, 9.6 µ g sFcγRIIB subgroup in the treatment group; tre-180 µ l, 14.4 µ g sFcγRIIB subgroup in the treatment group. * P<0.05; ** P<0.01; n=10 in each group. SLE, systemic lupus erythematosus; mFcγRIIB, membrane-bound type FcγRIIB; sFcγRIIB, soluble FcγRIIB.

Article Snippet: Serum FcγRIIB-IgG was assayed using rabbit anti-mouse FcγRIIB monoclonal antibody (cat. no. sc-365864; 1:2,000; Santa Cruz Biotechnology, Inc.) and HRP-labeled goat anti-mouse IgG antibody (cat. no. bs-0296G-HRP; 1:5,000; Beijing Bioss Biotechnology Co., Ltd.).

Techniques: Binding Assay, Control, Membrane

Journal: International Journal of Molecular Medicine

Article Title: Overexpression of FcγRIIB regulates downstream protein phosphorylation and suppresses B cell activation to ameliorate systemic lupus erythematosus

doi: 10.3892/ijmm.2020.4698

Figure Lengend Snippet: (A) The protein expression levels of BTK, p-BTK, Lyn, p-Lyn, SHIP and p-SHIP in MRL/lpr SLE mouse B cells were examined by western blot analysis, with β-actin as a loading control. (B and C) The phosphorylation and total protein levels of BTK, Lyn and SHIP were detected in B cells from MRL/lpr SLE mice after the infection of sFcγRIIB lentivirus, and the relative level for each phosphorylation-protein/each respective total protein was calculated. Lane 1: pre-control, control subgroup in the prevention group; lane 2: pre-60 µ l, 4.8 µ g sFcγRIIB subgroup in the prevention group; lane 3: pre-120 µ l, 9.6 µ g sFcγRIIB subgroup in the prevention group; lane 4: pre-180 µ l, 14.4 µ g sFcγRIIB subgroup in the prevention group; lane 5: tre-control, control subgroup in the treatment group; lane 6: tre-60 µ l, 4.8 µ g sFcγRIIB subgroup in the treatment group; lane 7: tre-120 µ l, 9.6 µ g sFcγRIIB subgroup in the treatment group; lane 8: tre-180 µ l, 14.4 µ g sFcγRIIB subgroup in the treatment group. * P<0.05; ** P<0.01; n=10 in each group. SLE, systemic lupus erythematosus; mFcγRIIB, membrane-bound type FcγRIIB; sFcγRIIB, soluble FcγRIIB; BTK, Bruton's tyrosine kinase; Lyn, Lyn proto-oncogene, Src family tyrosine kinase; DOK-1, docking protein 1; SHIP, inositol polyphosphate-5-phosphatase D.

Article Snippet: Serum FcγRIIB-IgG was assayed using rabbit anti-mouse FcγRIIB monoclonal antibody (cat. no. sc-365864; 1:2,000; Santa Cruz Biotechnology, Inc.) and HRP-labeled goat anti-mouse IgG antibody (cat. no. bs-0296G-HRP; 1:5,000; Beijing Bioss Biotechnology Co., Ltd.).

Techniques: Expressing, Western Blot, Control, Phospho-proteomics, Infection, Membrane

Journal: International Journal of Medical Sciences

Article Title: Serum transferrin predicts end-stage Renal Disease in Type 2 Diabetes Mellitus patients

doi: 10.7150/ijms.46259

Figure Lengend Snippet: Baseline clinical characteristics of individuals with and without ESRD

Article Snippet: Without washing, they were incubated with mouse monoclonal anti-transferrin antibody (sc365871, Santa Cruz, USA) at 4°C overnight and then incubated with an HRP-conjugated goat anti-mouse antibody (8125, CST, USA) at 37°C for 1 hour.

Techniques:

Journal: International Journal of Medical Sciences

Article Title: Serum transferrin predicts end-stage Renal Disease in Type 2 Diabetes Mellitus patients

doi: 10.7150/ijms.46259

Figure Lengend Snippet: Univariate ( a ) and multivariate ( b, c ) Cox proportional hazard models by serum iron status at the renal endpoint. Model 1: adjusted for age, sex, estimated glomerular filtration rate, proteinuria, hemoglobin, and HbA1c. Model 2: adjusted for the above plus pathological parameters including Renal Pathology Society diabetic nephropathy class, tubular atrophy and interstitial fibrosis, interstitial inflammation, arteriosclerosis, and arteriolar hyalinosis. Abbreviations: HR, hazard ratio; CI, confidence interval; TIBC, total iron-binding capacity; TSAT, transferrin saturation; Ref, reference.

Article Snippet: Without washing, they were incubated with mouse monoclonal anti-transferrin antibody (sc365871, Santa Cruz, USA) at 4°C overnight and then incubated with an HRP-conjugated goat anti-mouse antibody (8125, CST, USA) at 37°C for 1 hour.

Techniques: Filtration, Binding Assay

Journal: International Journal of Medical Sciences

Article Title: Serum transferrin predicts end-stage Renal Disease in Type 2 Diabetes Mellitus patients

doi: 10.7150/ijms.46259

Figure Lengend Snippet: Baseline clinical characteristics of individuals stratified by quartiles of baseline serum transferrin levels

Article Snippet: Without washing, they were incubated with mouse monoclonal anti-transferrin antibody (sc365871, Santa Cruz, USA) at 4°C overnight and then incubated with an HRP-conjugated goat anti-mouse antibody (8125, CST, USA) at 37°C for 1 hour.

Techniques:

Journal: International Journal of Medical Sciences

Article Title: Serum transferrin predicts end-stage Renal Disease in Type 2 Diabetes Mellitus patients

doi: 10.7150/ijms.46259

Figure Lengend Snippet: Pathological characteristics of individuals stratified by quartiles of baseline serum transferrin levels

Article Snippet: Without washing, they were incubated with mouse monoclonal anti-transferrin antibody (sc365871, Santa Cruz, USA) at 4°C overnight and then incubated with an HRP-conjugated goat anti-mouse antibody (8125, CST, USA) at 37°C for 1 hour.

Techniques:

Journal: International Journal of Medical Sciences

Article Title: Serum transferrin predicts end-stage Renal Disease in Type 2 Diabetes Mellitus patients

doi: 10.7150/ijms.46259

Figure Lengend Snippet: Transferrin and iron staining in kidney biopsy specimens. ( a ) Tissue transferrin (red arrows) by immunohistochemistry in normal kidney tissue, diabetic kidney tissue with microalbuminuria or macroalbuminuria. 200×, Bar=50 μm. ( b ) Quantification of the transferrin staining shows a significant increase in diabetic kidney tissue compared with normal kidney tissue (*p <0 001, compared with control group). Experiments were performed with ten participants for each group, and the results are expressed as mean ± SD. ( c ) Tissue iron deposition (black arrows) by Perl's staining in normal kidney tissue and diabetic kidney tissue. 200×, Bar=50 μm.

Article Snippet: Without washing, they were incubated with mouse monoclonal anti-transferrin antibody (sc365871, Santa Cruz, USA) at 4°C overnight and then incubated with an HRP-conjugated goat anti-mouse antibody (8125, CST, USA) at 37°C for 1 hour.

Techniques: Staining, Immunohistochemistry, Control

Journal: Cell Death & Disease

Article Title: TNFRSF receptor-specific antibody fusion proteins with targeting controlled FcγR-independent agonistic activity

doi: 10.1038/s41419-019-1456-x

Figure Lengend Snippet: a Domain architecture of TNFR2. b HeLa-TNFR2 cells were incubated in triplicates with anti-TNFR2 antibodies recognizing the indicated domain of TNFR2 along with HEK293 cells transfected with empty vector (EV) or a FcγR2B-encoding expression plasmid. One day later, cell supernatants were analyzed for IL8 production. Stimulation with a saturating concentration (200 ng/ml) of the highly potent TNFR2 agonist TNC-scTNF80 served as a positive control. c TNFR2-responsive HeLa-TNFR2 cells and Fn14-responsive WiDr cells were challenged in triplicates with the indicated anti-TNFRSF receptor antibodies and empty vector (EV) or FcγR2B transfected HEK293 cells which have no (TNFR2) or only low (Fn14) endogenous expression of the TNFRSF receptors studied and which only produce limited amounts of IL8. Next day, cell supernatants were analyzed for IL8 production as readout of TNFRSF receptor activation. d HeLa-TNFR2 cells were treated overnight in triplicates with HEK293 cells transfected with empty vector or expression plasmids encoding the indicated FcγR types and 1 µg/ml of IgG1, IgG2, IgG3, IgG4, mIgG1, and mIgG2A variants of the anti-TNFR2 antibody C4. Finally, IL8 production was quantified by ELISA

Article Snippet: Antibodies for evaluation of FcγR expression on THP-1 cells antibodies were from Santa Cruz Biotechnology, Santa Cruz, CA, USA (anti-CD16 PE-labeled mouse IgG1, #sc-19620; anti-CD64 PE-labeled mouse IgG1, #sc-1184; anti-CD32A/C mouse IgG1, #sc-373721; anti-CD32B mouse IgG1, #sc-365864) and Sigma, Saint Louis, MO, USA (anti-mouse IgG PE-labeled polyclonal goat, #P9670).

Techniques: Incubation, Transfection, Plasmid Preparation, Expressing, Concentration Assay, Positive Control, Activation Assay, Enzyme-linked Immunosorbent Assay

Journal: Cell Death & Disease

Article Title: TNFRSF receptor-specific antibody fusion proteins with targeting controlled FcγR-independent agonistic activity

doi: 10.1038/s41419-019-1456-x

Figure Lengend Snippet: a The affinities of the indicated TNFR2-specific mIgG1 antibodies were evaluated by binding studies with TNFR2-Fc-GpL and plastic immobilized antibodies. b Co-cultures of HeLa-TNFR2 cells and murine CD32B-expressing HEK293 cells were challenged overnight with the indicated concentrations of the TNFR2-specific mIgG1 antibodies. Finally, IL8 production was quantified by an ELISA

Article Snippet: Antibodies for evaluation of FcγR expression on THP-1 cells antibodies were from Santa Cruz Biotechnology, Santa Cruz, CA, USA (anti-CD16 PE-labeled mouse IgG1, #sc-19620; anti-CD64 PE-labeled mouse IgG1, #sc-1184; anti-CD32A/C mouse IgG1, #sc-373721; anti-CD32B mouse IgG1, #sc-365864) and Sigma, Saint Louis, MO, USA (anti-mouse IgG PE-labeled polyclonal goat, #P9670).

Techniques: Binding Assay, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Cell Death & Disease

Article Title: TNFRSF receptor-specific antibody fusion proteins with targeting controlled FcγR-independent agonistic activity

doi: 10.1038/s41419-019-1456-x

Figure Lengend Snippet: a Domain architecture and structural organization of C4-IgG1(N297A)-HC:scGITRL, C4-IgG1(N297A)-HC:sc(mu)4-1BBL, C4-IgG1(N297A)-HC:(mu)GITRL, and C4-IgG1(N297A)-HC:muIL2. b HeLa-TNFR2 cells and HEK293 transfectants expressing the indicated cytokine receptors were stimulated with 200 ng/ml of the various C4-IgG1(N297A) cytokine fusion proteins in the presence and absence of 1 µg/ml protein G. Next day, IL8 content of supernatants were determined by ELISA. c Co-cultures of HeLa-TNFR2 cells and the various HEK293 transfectants were stimulated as indicated with the C4-IgG1(N297A) cytokine fusion proteins and IL8 production was again determined the following day by ELISA

Article Snippet: Antibodies for evaluation of FcγR expression on THP-1 cells antibodies were from Santa Cruz Biotechnology, Santa Cruz, CA, USA (anti-CD16 PE-labeled mouse IgG1, #sc-19620; anti-CD64 PE-labeled mouse IgG1, #sc-1184; anti-CD32A/C mouse IgG1, #sc-373721; anti-CD32B mouse IgG1, #sc-365864) and Sigma, Saint Louis, MO, USA (anti-mouse IgG PE-labeled polyclonal goat, #P9670).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay

Journal: Cell Death & Disease

Article Title: TNFRSF receptor-specific antibody fusion proteins with targeting controlled FcγR-independent agonistic activity

doi: 10.1038/s41419-019-1456-x

Figure Lengend Snippet: a Domain architecture and structural organization of C4-IgG1(N297A)-HC:scFvCD19, C4-IgG1(N297A)-HC:scFvCD20, and C4-IgG1(N297A)- HC:scFvCD70. b Analysis of CD19, CD20, and CD70 expression of Jurkat and BJAB cells by FACS. c HeLa-TNFR2 cells and BJAB (CD19 + CD20 + CD70 + ) or Jurkat cells were cocultured and stimulated overnight with the indicated concentration of the various C4-IgG1(N297A) scFv fusion proteins. IL8 content of supernatants were determined by ELISA. d HeLa-TNFR2 cells alone or in combination with the indicated HEK293 transfectants were preincubated with 20 µM MLN4924 for 30 min and afterwards stimulated for the different times with C4-IgG1(N297A)-HC:scFvCD70 or TNC-sc(mu)TNF80, a potent TNFR2 agonist as a positive control. MLN4924 was used to prevent proteasomal IκBα degradation to rule out underestimation of IκBα phosphorylation. MLN4924 is an inhibitor which interferes with the activity of the E3 ligase complex responsible for K48 ubiquitination of IκBα

Article Snippet: Antibodies for evaluation of FcγR expression on THP-1 cells antibodies were from Santa Cruz Biotechnology, Santa Cruz, CA, USA (anti-CD16 PE-labeled mouse IgG1, #sc-19620; anti-CD64 PE-labeled mouse IgG1, #sc-1184; anti-CD32A/C mouse IgG1, #sc-373721; anti-CD32B mouse IgG1, #sc-365864) and Sigma, Saint Louis, MO, USA (anti-mouse IgG PE-labeled polyclonal goat, #P9670).

Techniques: Expressing, Concentration Assay, Enzyme-linked Immunosorbent Assay, Positive Control, Activity Assay

Journal: Cell Death & Disease

Article Title: TNFRSF receptor-specific antibody fusion proteins with targeting controlled FcγR-independent agonistic activity

doi: 10.1038/s41419-019-1456-x

Figure Lengend Snippet: a CD40- (HT1080-CD40), 4-1BB- (HT1080-4-1BB), CD95- (HT1080), and Fn14-responsible cells (HeLa) were cocultured with HEK293-EV and HEK293-CD20 cells. Co-cultures were stimulated with the indicated concentrations of anti-4-1BB-IgG1(N297A)-HC:scFvCD20, anti-CD95-IgG1(N297A)-HC:scFvCD20, anti-Fn14-IgG1(N297A)-HC:scFvCD20, and anti-CD40-IgG1(N297A)-HC:scFvCD20. Next day, TNFRSF receptor activation was evaluated by measuring IL8 production. The CD95-specific antibody was applied in the presence of 20 µM ZVAD to prevent apoptosis. b Co-cultures were performed as described in “A” with the difference that HEK293-EV cells have been replaced by Jurkat cells and HEK293-CD20 cells by BJAB cells. c Co-cultures of Jurkat or BJAB cells with the indicated TNFRSF receptor-responsive cell lines were stimulated overnight in the presence and absence of 50 µg/ml anti-CD20-IgG1 with 100 ng/ml of the various IgG1(N297A)-HC:scFvCD20 fusion proteins and IL8 production was finally quantified by ELISA. In case of anti-CD40-IgG1(N297A)-HC:scFvCD20 cells were treated with only 20 ng/ml

Article Snippet: Antibodies for evaluation of FcγR expression on THP-1 cells antibodies were from Santa Cruz Biotechnology, Santa Cruz, CA, USA (anti-CD16 PE-labeled mouse IgG1, #sc-19620; anti-CD64 PE-labeled mouse IgG1, #sc-1184; anti-CD32A/C mouse IgG1, #sc-373721; anti-CD32B mouse IgG1, #sc-365864) and Sigma, Saint Louis, MO, USA (anti-mouse IgG PE-labeled polyclonal goat, #P9670).

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay