Journal:

Article Title: Expression of β 1 Integrins in Sensory Neurons of the Dorsal Root Ganglion and Their Functions in Neurite Outgrowth on Two Laminin Isoforms

doi:

Figure Lengend Snippet: Differential effects of integrin-α-subunit–specific antibodies on human DRG neurite outgrowth on laminin isoforms AeB1eB2e and AmB1eB2e: fluorescent micrographs of postfixation, Dil-labeled human DRG neurons cultured 16 hr on theAeB1eB2e (A, C, E, G) or the AmB1eB2e (B, D, F, H) isoform of laminin in the absence of antibodies (A, B) or in the presence of mAb anti-β1 (C, D), anti-α1β1 (E, F), or anti-α3β1 (G, H). Antibody concentrations used are given in Materials and Methods. Scale bar, 50 μm.

Article Snippet: All of the monoclonal antibodies (mAbs) were previously characterized as function-blocking antibodies. mAbs PIE6 (anti- α 2 β 1; IgG1), PIB5 (anti- α 3 β 1; IgG1), and PID6 (anti- α 5 β 1; IgG3) have been characterized ( Wayner and Carter, 1987 ) and were purchased as ascites from Telios Inc. (La Jolla, CA). mAbs A2B2 (anti- β 1; IgG1) and S2G3 (anti- α 1 β 1; IgM) have been characterized previously ( Hall et al., 1990 ).

Techniques: Labeling, Cell Culture

Journal:

Article Title: Expression of β 1 Integrins in Sensory Neurons of the Dorsal Root Ganglion and Their Functions in Neurite Outgrowth on Two Laminin Isoforms

doi:

Figure Lengend Snippet: Effect of anti-α1β1 on PC12 cell attachment to laminins AeB1eB2e or AmB1eB2e. a, PC 12 cell attachment to various concentrations of AeB1eB2e laminin in the absence (open circles) or in the presence (solid circles) of anti-α1 (mAb 3A3, 10 μg/ml). b, PC 12 cell attachment to various concentrations of AmB1eB2e in the absence (open circles) or in the presence (solid circles) of anti-α1 (mAb 3A3, 10 μg/ml).

Article Snippet: All of the monoclonal antibodies (mAbs) were previously characterized as function-blocking antibodies. mAbs PIE6 (anti- α 2 β 1; IgG1), PIB5 (anti- α 3 β 1; IgG1), and PID6 (anti- α 5 β 1; IgG3) have been characterized ( Wayner and Carter, 1987 ) and were purchased as ascites from Telios Inc. (La Jolla, CA). mAbs A2B2 (anti- β 1; IgG1) and S2G3 (anti- α 1 β 1; IgM) have been characterized previously ( Hall et al., 1990 ).

Techniques: Cell Attachment Assay

Journal: British Journal of Cancer

Article Title: Downregulation of urokinase plasminogen activator receptor expression inhibits Erk signalling with concomitant suppression of invasiveness due to loss of uPAR– β 1 integrin complex in colon cancer cells

doi: 10.1038/sj.bjc.6601098

Figure Lengend Snippet: Characterisation of HCT116 cells. ( A ) Southern blot analyses of genomic DNAs isolated from transfected and untransfected HCT116 cells. Lane 1, WT HCT116 cells; lane 2, mock-transfected HCT116 cells; and lane 3 is 5′A/S HCT116 cells. ( B ) Flow-cytometric analyses of uPAR and β 1 integrin in HCT116 cell lines. The median intensity of fluorescence (MIF, arbitary units, log scale) was measured. Results are representative of three independent experiments.

Article Snippet: The monoclonal antibodies against integrin α 1 (FB12), α 2 (PIE6), α 3 (PIB5), α 4 (PIH4), α 5 (PID6), α 6 (CLB-701), α v (LM 142) and β 1 (P5D2), were from Chemicon International (CA, USA).

Techniques: Southern Blot, Isolation, Transfection, Fluorescence

Journal: British Journal of Cancer

Article Title: Downregulation of urokinase plasminogen activator receptor expression inhibits Erk signalling with concomitant suppression of invasiveness due to loss of uPAR– β 1 integrin complex in colon cancer cells

doi: 10.1038/sj.bjc.6601098

Figure Lengend Snippet: Cell surface expression at α integrin subunits in mock and A/S HCT116 cell lines

Article Snippet: The monoclonal antibodies against integrin α 1 (FB12), α 2 (PIE6), α 3 (PIB5), α 4 (PIH4), α 5 (PID6), α 6 (CLB-701), α v (LM 142) and β 1 (P5D2), were from Chemicon International (CA, USA).

Techniques: Expressing

Journal: British Journal of Cancer

Article Title: Downregulation of urokinase plasminogen activator receptor expression inhibits Erk signalling with concomitant suppression of invasiveness due to loss of uPAR– β 1 integrin complex in colon cancer cells

doi: 10.1038/sj.bjc.6601098

Figure Lengend Snippet: Coimmunoprecipitation of β 1 integrin and uPAR. Mock- and A/S-transfected HCT116 cells were lysed in Triton X-100 buffer (see Materials and Methods). Cell protein (500 μ g) was mixed with anti- β 1 antibody (PD52) or anti-uPAR antibody (3936) or isotype-matched mouse IgG and the resulting immunoprecipitates were analysed by Western blotting with ( A ) anti- β 1-integrin antibody (PD52) or ( B ) anti-uPAR antibody (3936). ( C ) Cells were surface biotinylated and cell extracts were subjected to immunoprecipitation with anti- β 1 antibody or with isotype-matched IgG antibodies and analysed by streptavidin–HRP binding to biotinylated proteins after SDS–PAGE and transfer to nitrocellulose membranes. ( D and E ) Mock-transfected cell lysates were immunodepleted (ID)of β 1 after five rounds of sequential β 1 and uPAR immunoprecipitation. Cell lysates were resolved by 10% SDS–PAGE followed by blotting with ( D ) anti-uPAR and ( E ) anti- β 1 integrin antibody. The experiments were repeated at least three times.

Article Snippet: The monoclonal antibodies against integrin α 1 (FB12), α 2 (PIE6), α 3 (PIB5), α 4 (PIH4), α 5 (PID6), α 6 (CLB-701), α v (LM 142) and β 1 (P5D2), were from Chemicon International (CA, USA).

Techniques: Transfection, Western Blot, Immunoprecipitation, Binding Assay, SDS Page

Journal: British Journal of Cancer

Article Title: Downregulation of urokinase plasminogen activator receptor expression inhibits Erk signalling with concomitant suppression of invasiveness due to loss of uPAR– β 1 integrin complex in colon cancer cells

doi: 10.1038/sj.bjc.6601098

Figure Lengend Snippet: uPAR/ β 1 integrin complex and the affect of P25 peptide. ( A ) P25 peptide but not scrambled peptide (Scp) disrupts uPAR/ β 1 integrin complex in mock-transfected HCT116 cells. Cells were treated for 16 h with P25 peptide (100 μ M ) and Scp (100 μM). Immunoprecipitates of uPAR were prepared and coimmunoprecipitated β 1 integrin band was analysed. ( B ) Migration/invasion of HCT116 mock cells in the presence of P25 peptide and its scrambled analogue (100 μ M each). ( C ) Plasminogen-dependent matrix degradation of HCT116 mock cells in the presence of P25 peptide and its scrambled analogue (100 μ M each). Results for both are shown as mean + S.E.M. of three different experiments performed in triplicate ( * P <0.001, compared to control cells in the presence of Plg). ( D ) Effects of P25 peptide and its scrambled analogue on the secretion and activation of pro-MMP-2/MMP-9. Conditioned medium in the presence and absence of Plg, P25 peptide and Scp (100 μ M each) was prepared as described in the Materials and Methods section. The samples were analysed by equal protein loading by gelatin-zymography for the activation of pro-MMP-2/MMP-9. Quantification of MMP secretion in the tumour-conditioned medium was performed by densitometry and the results are expressed as peak OD. Results are representative of one experiment. The experiment was repeated three times. ( E ) Effect of P25 peptide and its scrambled analogue (100 μ M each) on uPA-induced activation of Erk in mock-transfected HCT116 cells. Subconfluent cultures of mock-transfected HCT116 cells were serum starved for 24 h, acid stripped for 1 min and incubated with uPA (20 n M ) for 30 min. The level of phospho Erk1/2 was determined by Western blot using equal protein loading. Quantification of phospho Erk1/2 expression was performed by densitometry and expressed as peak OD. Results are representative of one experiment. The experiment was repeated three times.

Article Snippet: The monoclonal antibodies against integrin α 1 (FB12), α 2 (PIE6), α 3 (PIB5), α 4 (PIH4), α 5 (PID6), α 6 (CLB-701), α v (LM 142) and β 1 (P5D2), were from Chemicon International (CA, USA).

Techniques: Transfection, Migration, Activation Assay, Zymography, Incubation, Western Blot, Expressing