Journal: Frontiers in Immunology

Article Title: Complement Protein C1q Interacts with DC-SIGN via Its Globular Domain and Thus May Interfere with HIV-1 Transmission

doi: 10.3389/fimmu.2016.00600

Figure Lengend Snippet: Interaction of C1q, ghA, ghB, and ghC with DC-SIGN tetramer and monomer . (A) Microtiter wells coated with different concentrations (5, 2.5, 1.25, 0.625 µg/well) of DC-SIGN tetramer or monomer were probed with 2 µg/well of C1q. Bound C1q was detected with anti-C1q polyclonal antibodies (1:1,000 in PBS) and Protein A HRP conjugate (1:1,000 in PBS). BSA was used as a negative control protein. (B) Binding of ghA, ghB, and ghC to DC-SIGN tetramer and (C) DC-SIGN monomer involved coating a range of concentrations of the respective proteins on microtiter wells, which were then incubated with a fixed concentration of ghA, ghB, ghC, and MBP (2.5 µg/well in 5 mM CaCl 2 buffer) at 37°C. Binding was detected using anti-MBP monoclonal antibodies (1:5,000 in PBS) and then rabbit anti-mouse IgG-HRP (1:5,000 in PBS). (D) Far western blot to show DC-SIGN tetramer binding to membrane-bound ghA, ghB, and ghC: 15 µg of ghA, ghB, and ghC (BSA and MBP as negative control proteins) were run on a 12% SDS-PAGE gel and then transferred on to nitrocellulose membrane. The blot was incubated with 50 µg of DC-SIGN in PBS overnight at room temperature. The bound DC-SIGN protein was detected using anti-DC-SIGN polyclonal antibodies and Protein A HRP conjugate. Bands were developed using diaminobenzidine tablets dissolved in water.

Article Snippet: The blot was washed three times for 10 min each in PBS containing 0.05% Tween 20 and then incubated with anti-DC-SIGN (1:1,000) polyclonal antibody (ProSci) in 1% non-fat milk (2 h at 37°C).

Techniques: Negative Control, Binding Assay, Incubation, Concentration Assay, Bioprocessing, Far Western Blot, Membrane, SDS Page

Journal: Frontiers in Immunology

Article Title: Complement Protein C1q Interacts with DC-SIGN via Its Globular Domain and Thus May Interfere with HIV-1 Transmission

doi: 10.3389/fimmu.2016.00600

Figure Lengend Snippet: Interaction of C1q, ghA, ghB, and ghC with DC-SIGNR tetramer and monomer . (A) ELISA to examine binding of C1q to DC-SIGNR tetramer and SIGN-R monomer: DC-SIGNR tetramer or monomer were coated at different concentrations, followed by addition of 2 µg/well of C1q. Bound C1q was probed with anti-C1q polyclonal antibodies (1:1,000 in PBS) and Protein A HRP (1:1,000 in PBS), and the color was developed using o-phenylenediamine dihydrochloride. (B) Binding of ghA, ghB, and ghC to DC-SIGNR tetramer and (C) SIGN-R monomer: different concentrations of DC-SIGNR tetramer (B) and DC-SIGNR monomer (C) were coated on microtiter wells in carbonate buffer and incubated overnight at 4°C and then incubated with ghA, ghB, ghC, and MBP (2.5 µg/well in 5 mM CaCl 2 buffer). Binding was detected using anti-MBP monoclonal antibody and rabbit anti-mouse IgG-HRP conjugate.

Article Snippet: The blot was washed three times for 10 min each in PBS containing 0.05% Tween 20 and then incubated with anti-DC-SIGN (1:1,000) polyclonal antibody (ProSci) in 1% non-fat milk (2 h at 37°C).

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

Journal: Frontiers in Immunology

Article Title: Complement Protein C1q Interacts with DC-SIGN via Its Globular Domain and Thus May Interfere with HIV-1 Transmission

doi: 10.3389/fimmu.2016.00600

Figure Lengend Snippet: Competitive inhibition of DC-SIGN: HIV-1 gp120 interaction by globular head modules and gC1qR . (A) ELISA to assess whether gC1qR and ghB directly compete for the same binding site on DC-SIGN: DC-SIGN was coated at 5 µg/well overnight at 4°C. Wells were blocked with 2% BSA in PBS for 2 h at 37°C. gC1qR (5 µg/well) and different concentrations of ghB (5, 2.5, 1.25, 0.625 µg/well) were added in buffer containing 5 mM CaCl 2 . Incubation was carried out at 37°C for 1.5 h and 4°C for 1.5 h. Following repeated washes, bound gC1qR was probed using rabbit anti-gC1qR polyclonal antibodies (1:1,000) and Protein A-HRP (1:1,000). Color was developed using o -phenylenediamine dihydrochloride substrate; (B) competition between DC-SIGN tetramer and C1q globular head modules to bind solid-phase gp120. Microtiter wells were coated with 250 ng of gp120. Various concentrations of ghA, ghB, ghC, and C1q and constant 2.5 µg/mL of DC-SIGN were incubated at 37°C for 1 h and then at 4°C for 1 h. The binding of DC-SIGN to gp120 in the presence of globular heads or C1q was detected using rabbit anti-DC antibody (1:500), probed with Protein A HRP (1:5,000). DC-SIGN alone binding to gp120 was used as 100%.

Article Snippet: The blot was washed three times for 10 min each in PBS containing 0.05% Tween 20 and then incubated with anti-DC-SIGN (1:1,000) polyclonal antibody (ProSci) in 1% non-fat milk (2 h at 37°C).

Techniques: Inhibition, Enzyme-linked Immunosorbent Assay, Binding Assay, Incubation

Journal: Advanced Science

Article Title: Identification and Characterization of the Wilms Tumor Cancer Stem Cell

doi: 10.1002/advs.202206787

Figure Lengend Snippet: List of antibodies used in the experimental procedures (IF: immunofluorescence, WB: Western blot)

Article Snippet: CLEC4M polyclonal antibody , Proteintech Group # 22003 , 1:50 IF.

Techniques: Immunofluorescence, Western Blot, In Vivo