Journal: The Journal of Biological Chemistry

Article Title: Flanking V and J Sequences of Complementary Determining Region 3 of T Cell Receptor (TCR) δ1 (CDR3δ1) Determine the Structure and Function of TCRγ4δ1 *

doi: 10.1074/jbc.M111.239624

Figure Lengend Snippet: Binding activity of CDR3δ1 and its mutant peptides to tumor cells/tissues or ligand MICA. A , shown are schema of the amino acid sequences of GTM and its V/D/J mutants. GTM , GTMVm , GTMDm , and GTMJm represent the wild type and mutant peptides in V, D, and J region of a CDR3δ1, respectively. B , shown are flow cytometry profiles of the binding of GTM or its mutant peptides to various tumor cell lines BGC823, G401, GLC-82, HT29, and SKOV3. After incubation with biotinylated peptides, tested cells were stained with FITC-streptavidin and then analyzed by flow cytometry. Data are representative of three independent experiments. C , shown are confocal images of the binding of GTM peptide or its mutant peptides to various tumor cells. After incubation with the peptides, tested cells were stained with FITC-streptavidin and then analyzed by laser scanning confocal microscopy. Confocal images are one representative of at least three independent experiments. D , binding of GTM peptide and its mutants to primary tumor specimens was analyzed by immunohistochemistry. CH3 peptide was served as negative control. Binding was visualized using diaminobenzidine as the substrate ( brown ) (×100). G , gastric cancer; R , renal carcinoma; L , lung carcinoma; C , colon carcinoma; O , ovarian cancer. E , binding of GTM peptide and its mutants to MICA was analyzed by ELISA. Data shown are the mean of three independent experiments. F , binding of GTM peptide and its mutants to MICA was analyzed by SPR. The K D values are shown in the figure.

Article Snippet: Fluorescein isothiocyanate (FITC)-conjugated streptavidin (Pierce) or FITC-conjugated goat anti-human IgG antibody was then added and incubated for 30 min at 4 °C.

Techniques: Binding Assay, Activity Assay, Mutagenesis, Flow Cytometry, Incubation, Staining, Confocal Microscopy, Immunohistochemistry, Negative Control, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Biological Chemistry

Article Title: Flanking V and J Sequences of Complementary Determining Region 3 of T Cell Receptor (TCR) δ1 (CDR3δ1) Determine the Structure and Function of TCRγ4δ1 *

doi: 10.1074/jbc.M111.239624

Figure Lengend Snippet: Specific binding of TCR γ4-Fc/δ1(GTM)-Fc fusion protein and its mutants to tumor cells or tissues and antigen MICA. A , the expression of γ4-Fc/δ1(GTM)-Fc fusion protein and its mutants was analyzed by SDS-PAGE and Western blotting. Two subunits with different M r of the four fusion proteins are shown by reduced PAGE ( upper left ). TCRγ4-Fc chain ( upper bands ) ran slightly higher than TCRδ1-Fc ( lower bands ). Intact γ4-Fc/δ1-Fc dimmers are shown by non-reduced PAGE ( lower left ). Lane 1 , γ4-Fc/δ1(GTM)-Fc; lane 2 , γ4-Fc/δ1(GTMVm)-Fc; lane 3 , γ4-Fc/δ1(GTMDm)-Fc; lane 4 , γ4-Fc/δ1(GTMJm)-Fc; lane M , protein marker. We further characterized the fusion proteins by Western blotting using anti-Vδ, anti-Vγ, and anti-human IgG Fc as primary antibody ( right panels ). B , binding of TCRγ4-Fc/δ1(GTM)-Fc fusion protein and its V, D, and J mutants to tumor cells was analyzed by flow cytometry. After incubation with fusion proteins, tested cells were stained with FITC-conjugated goat anti-human IgG and then analyzed by flow cytometry. Data are representative of three independent experiments. C , binding of γ4-Fc/δ1(GTM)-Fc and its V, D, and J mutants to tumor cells was analyzed by confocal microscopy. After incubation with fusion proteins, cells were stained with FITC-conjugated goat anti-human IgG and followed by laser scanning confocal microscopic analysis. Confocal images are representative of three independent experiments. D , binding of γ4-Fc/δ1(GTM)-Fc protein and its V, D, and J mutants to primary tumor specimens was analyzed by immunohistochemistry. Human IgG Fc was used as a negative control. Binding was visualized using diaminobenzidine as the substrate ( brown ) (×100). G , gastric cancer; R , renal carcinoma; L , lung carcinoma; C , colon carcinoma; O , ovarian cancer. E , shown is ELISA analysis of the binding of γ4-Fc/δ1(GTM)-Fc and its mutants to antigen MICA. Data shown are the mean of three independent experiments. F , shown is SPR analysis of the binding of γ4-Fc/δ1(GTM)-Fc and its mutants to MICA antigen. The K D values are shown in the figure.

Article Snippet: Fluorescein isothiocyanate (FITC)-conjugated streptavidin (Pierce) or FITC-conjugated goat anti-human IgG antibody was then added and incubated for 30 min at 4 °C.

Techniques: Binding Assay, Expressing, SDS Page, Western Blot, Marker, Flow Cytometry, Incubation, Staining, Confocal Microscopy, Immunohistochemistry, Negative Control, Enzyme-linked Immunosorbent Assay