Journal: Scientific Reports

Article Title: Heparan sulfate proteoglycans (HSPGs) and chondroitin sulfate proteoglycans (CSPGs) function as endocytic receptors for an internalizing anti-nucleic acid antibody

doi: 10.1038/s41598-017-14793-z

Figure Lengend Snippet: CD44 and BCAN, representative CSPGs, act as endocytic receptors for 3D8 scFv on HeLa and HEK293 cells. ( a ) Schematic diagrams of CD44 and BCAN, which are expressed on the cell surface, and their recombinants, which are expressed intracellularly (controls). SP, signal peptide; Δ, deletion; TM, transmembrane domain; CD, cytoplasmic domain; GPI, GPI anchor; GFP, green fluorescent protein; HA, HA tag. ( b – e ) Confocal microscopy. HeLa cells were transfected with plasmids encoding CD44-GFP ( b ) and HA-BCAN ( d ), which are expressed on the cell surface, or with plasmids encoding CD44ΔTM-GFP ( c ) and BCAN-ΔGPI-GFP ( e ), which are expressed intracellularly. At 18 h post-transfection, cells were incubated with 10 μM 3D8 scFv under the specified conditions. After fixation and permeabilization, cells transfected with plasmids containing the GFP gene were incubated with a rabbit anti-3D8 scFv antibody, followed by a TRITC-conjugated goat anti-rabbit IgG antibody. Cells transfected with the plasmid encoding the HA-BCAN gene were incubated with a rabbit anti-3D8 scFv antibody and a mouse anti-HA antibody, which were detected by TRITC-conjugated goat anti-rabbit IgG and Alexa Fluor 488-conjugated goat anti-mouse IgG, respectively. Nuclei were stained with Hoechst 33342 (blue). Bar , 10 μm. ( f , g ) Cell surface expression of endogenous CD44 and BCAN was examined by flow cytometry. ( h,i ) Co-immunoprecipitation. HeLa ( h ) and HEK293 cells ( i ) were transfected with plasmids encoding CD44-GFP and HA-BCAN and 24 h later treated with 5 μM 3D8 scFv for 6 h at 37 °C. Cells were collected and lysed for co-immunoprecipitation assays. Co-immunoprecipitation was performed using an anti-GFP antibody, Samples were analyzed by immunoblotting with antibodies specific for CD44 and the His tag (left panels of h, i). Samples from the co-immunoprecipitation performed with the anti-HA antibody, were analyzed by immunoblotting with antibodies specific for the HA tag and the His tag (right panels of h, i). Cells not treated with 3D8 scFv protein were used as a negative control. Proteins from the extract (Input; 10%) and pulled-down fractions (IP) were analyzed by immunoblotting. Asterisks denote endogenous CD44.

Article Snippet: Over-expression of HA-BCAN in pgsD-677 was detected with mouse anti-BCAN IgG (Abnova, cat# H00063827-B01P) and AlexaFluor 488-goat anti-mouse IgG (Invitrogen, cat# A10680).

Techniques: Confocal Microscopy, Transfection, Incubation, Plasmid Preparation, Staining, Expressing, Flow Cytometry, Immunoprecipitation, Western Blot, Negative Control

Journal: Scientific Reports

Article Title: Heparan sulfate proteoglycans (HSPGs) and chondroitin sulfate proteoglycans (CSPGs) function as endocytic receptors for an internalizing anti-nucleic acid antibody

doi: 10.1038/s41598-017-14793-z

Figure Lengend Snippet: 3D8 scFv prefers HS chains over CS and DS chains for binding and cellular internalization. ( a ) Competitive ELISA to determine the binding preference of 3D8 scFv for GAG chains. ELISA plates coated with 10 μg/ml soluble HS, CS-A, CS-C, or DS were incubated for 1 h at RT with 1:1 (v/v) mixtures of 3D8 scFv-pA (20 μg/ml) and each competitor (20 μg/ml), followed by detection of bound 3D8 scFv. ( b , c ) Competitive ELISA to compare the binding preference of 3D8 scFv between HS and CS. ELISA plates coated with 10 μg/ml of soluble CS-A, heparin ( b ), or 2 μg/ml of pcDNA3.1 plasmid DNA ( c ), were incubated with 1:1 (v/v) mixtures of 3D8 scFv-pA (20 μg/ml) and various concentrations (1–100 μg/ml) of competitors (heparin and CS-A, respectively). Binding of 3D8 scFv-pA to each antigen in the presence of competitors was determined using rabbit IgG, followed by AP-conjugated anti-rabbit IgG. Data represent the mean ± S.E. of triplicate wells and are representative of three independent experiments ( a–c ). ( d ) Flow cytometry to examine expression levels of endogenous HSPGs and CSPGs on CHO-K1 cell surface using antibodies specific for HSGPs and CSPGs. ( e , f ) Flow cytometry to examine internalization of 3D8 scFv in the presence of soluble GAGs. CHO-K1 ( e ) and HeLa cells ( f ) were incubated for 6 h at 37 °C with mixtures of 3D8 scFv (10 μM) and each soluble GAG (at the specified concentrations) ( g ) Flow cytometry to examine cell surface expression of HSPGs and CSPGs on CHO-K1 cells after enzyme treatment for 1 h at 37 °C (left panel) and the amount of internalized 3D8 scFv after enzyme treatment for 6 h at 37 °C (right panel). ( h – j ) Flow cytometry to detect internalization of 3D8 scFv by CHO cell lines treated with 3D8 scFv (5 μM) for 0.5–24 h at 37 °C (H), expression of CD44-GFP and HA-BCAN in pgsD-677 cells transfected with plasmids (I), and internalized 3D8 scFv in transfected pgsD-677 cells ( j ). A minimum of 10,000 cells per sample were analyzed. MFI, Mean Fluorescence Intensity. Data are expressed as the mean ± standard error of three independent experiments. All p values were calculated using a two-tailed Student’s t test. Statistical significance is indicated on the graphs (** p < 0.01; *** p < 0.001).

Article Snippet: Over-expression of HA-BCAN in pgsD-677 was detected with mouse anti-BCAN IgG (Abnova, cat# H00063827-B01P) and AlexaFluor 488-goat anti-mouse IgG (Invitrogen, cat# A10680).

Techniques: Binding Assay, Competitive ELISA, Enzyme-linked Immunosorbent Assay, Incubation, Plasmid Preparation, Flow Cytometry, Expressing, Transfection, Fluorescence, Two Tailed Test