Journal:

Article Title: Epitope Mapping of Function-blocking Monoclonal Antibody CM6 Suggests a "Weak" Integrin Binding Site on the Laminin-332 LG2 Domain

doi: 10.1002/jcp.22107

Figure Lengend Snippet: (A) Sequence alignment of human and rat laminin α3 LG2 module. The rat laminin α3 LG2 sequence was aligned to that of human laminin α3 LG2. There is a difference of total 29 amino acid residues between them. To delimitate the epitope of CM6, a total of 17 mutants were generated by replacing rat amino acid residues for human amino acid residues within the rat laminin α3 LG2 module using a site directed mutagenesis method. (B) In site-directed mutagenesis, pGEX-2T LG2 vector (which encodes rat laminin α3 LG2 module) was used as a template to generate 17 mutant LG2 proteins. Wild type and 17 mutant LG2 proteins were purified by the Nickel agarose beads. Purified recombinant wild type (lane 1) and 17 mutant LG2 proteins (lanes 2–17) were run on 12 % gel and separated gel was immunoblotted by CM6 mAb at the diluted ratio of 1: 200 to identify the epitope sequences. Antibody against GST was also used as a loading control. Out of a total of 17 mutants expressed, only 2 mutants (NE→DV, lane 14 and RSVR→QNTI, lane15) were negative against CM6 antibody. (C) To determine the specificity of CM6 binding, 96-well microplates were coated with Ln-332 (1 μg/ml), GST (50 μg/ml), rLG2 (50 μg/ml), or a synthetic peptide: NERSVR, DVQNTI, or GLLFFA (100 μg/ml) at 4°C overnight. CM6 mAb was added as a primary antibody to wells (50 μg/ml) and incubated for 2 h at RT. Anti-mouse IgG conjugated with HRP was added as a secondary antibody to well and incubated for 1 h at RT. Bound antibody was detected by measuring O.D. (450 nm). CM6 bound the synthetic peptide NERSVR similarly to intact Ln-332 (N=2, in triplicate; p=0.394) and rLG2 module (N=2, in triplicate; p=0.240). In contrast, compared to Ln-332, CM6 had almost no reactivity to the human sequence DVQNTI (N=2, in triplicate; p=0.002*) or the rat LG2 module sequence GLLFFA (N=2, in triplicate; p=0.002*), both of which resulted in measurements similar to antibody on GST or PBS.

Article Snippet: Sterile 96-well microplates were coated with Ln-332 (1 μg/ml), GST (50 μg/ml), rLG2 (50 μg/ml), or synthetic peptides: NERSVR, DVQNTI, or GLLFFA (100 μg/ml, Biopeptide Co. Inc., San Diego, CA) at 4°C overnight.

Techniques: Sequencing, Generated, Mutagenesis, Plasmid Preparation, Purification, Recombinant, Control, Binding Assay, Incubation

Journal:

Article Title: Epitope Mapping of Function-blocking Monoclonal Antibody CM6 Suggests a "Weak" Integrin Binding Site on the Laminin-332 LG2 Domain

doi: 10.1002/jcp.22107

Figure Lengend Snippet: (A) 96-well microplates were coated with Ln-332 or collagen-I at 4°C overnight. MCF10A cells were labeled with Celltracker™ green CMFDA in PBS (labeling solution) and incubated for 30 min at 37°C. After a washing step, cells were seeded in microplates (5000 cells in 100 μl per well), and allowed to incubate (and spread) for ~7 h, imaging every 6 min using a BD Pathways 855 Bioimager. Normal mouse IgG, CM6, or TR1 were also added where indicated. Representative images from each treatment are shown. Scale bar is equal to 50 μm. (B) Box-and-whisker plots representing mean cell speed (bold horizontal line), 25th and 75th quartiles (box), and 95% confidence intervals (whiskers) are shown (N=70–750). MCF10A cells treated with CM6 exhibited significantly reduced spreading when plated on Ln-332 (p<0.001), but not on collagen-I (p=0.118). In contrast, non-function-blocking antibody TR1 had almost no affect on spreading (p=0.984).

Article Snippet: Sterile 96-well microplates were coated with Ln-332 (1 μg/ml), GST (50 μg/ml), rLG2 (50 μg/ml), or synthetic peptides: NERSVR, DVQNTI, or GLLFFA (100 μg/ml, Biopeptide Co. Inc., San Diego, CA) at 4°C overnight.

Techniques: Labeling, Incubation, Imaging, Whisker Assay, Blocking Assay

Journal:

Article Title: Epitope Mapping of Function-blocking Monoclonal Antibody CM6 Suggests a "Weak" Integrin Binding Site on the Laminin-332 LG2 Domain

doi: 10.1002/jcp.22107

Figure Lengend Snippet: (A) 96-well microplates were coated with Ln-332 (1 μg/ml), GST (50 μg/ml), rLG2 (50 μg/ml), rLG3 (50 μg/ml) at 4°C overnight. MCF10A cells were added to wells (5000/well) and incubated for 5 h at 37°C in a humidified, 5% CO2, 95% air atmosphere. After incubation, attached cells were fixed and stained with Hoechst (blue) and phalloidin (green) in PBS containing 3% BSA for 2 h at RT. Cell spreading was quantified by manually counting spread cells in five representative microscope fields. (B) CM6 significantly reduced cell spreading on Ln-332 (N=5; p=0.008) and rLG2 (N=5; p=0.008). In contrast, CM6 had little effect on cell spreading on rLG3 module (N=5; p=0.222) or GST (N=5; p=0.095).

Article Snippet: Sterile 96-well microplates were coated with Ln-332 (1 μg/ml), GST (50 μg/ml), rLG2 (50 μg/ml), or synthetic peptides: NERSVR, DVQNTI, or GLLFFA (100 μg/ml, Biopeptide Co. Inc., San Diego, CA) at 4°C overnight.

Techniques: Incubation, Staining, Microscopy