pid6 antibody  (Telios Pharmaceuticals)

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