Journal: Molecular Pharmacology

Article Title: Bradykinin B 2 Receptor Interacts with Integrin ?5?1 to Transactivate Epidermal Growth Factor Receptor in Kidney Cells

doi: 10.1124/mol.110.064840

Figure Lengend Snippet: Involvement of integrins in BK-induced ERK phosphorylation. Quiescent mIMCD-3 cells were pretreated for 1 h either with 28 μM control or with cyclic RGD peptides before stimulation with 100 nM BK (A) or 1 ng/ml EGF (C) for 5 min. Quiescent mIMCD-3 cells were pretreated for 2 h with 0.1 mg/ml neutralizing anti-integrin antibodies before stimulation with 100 nM BK (B) or 1 ng/ml EGF (D) for 5 min. Control samples were preincubated with 0.1 mg/ml normal rabbit IgG. ERK phosphorylation was measured as described under Materials and Methods. Bars represent intensities of phospho-ERK bands relative to total ERK expressed as fold of basal (cells treated with vehicle). Experiments were performed three times in duplicate. Data are presented as mean + S.E.M. ***, p < 0.001..

Article Snippet: Antibodies against integrin α2, α5, α6, β6, and β1 subunits, blocking antibodies against α3, αV, and β3 integrins, MMP-8 siRNA, MMP-13 siRNA, integrin α2 siRNA, integrin α5 siRNA, integrin β1 siRNA, and control siRNA were from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques:

Journal: Molecular Pharmacology

Article Title: Bradykinin B 2 Receptor Interacts with Integrin ?5?1 to Transactivate Epidermal Growth Factor Receptor in Kidney Cells

doi: 10.1124/mol.110.064840

Figure Lengend Snippet: mIMCD-3 cells express mRNA for α and β integrin subunits The expression of mRNA was assessed by the Oligo GEArray as described under Materials and Methods . Proteins for which we were able to detect message are shown in bold

Article Snippet: Antibodies against integrin α2, α5, α6, β6, and β1 subunits, blocking antibodies against α3, αV, and β3 integrins, MMP-8 siRNA, MMP-13 siRNA, integrin α2 siRNA, integrin α5 siRNA, integrin β1 siRNA, and control siRNA were from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Expressing

Journal: Molecular Pharmacology

Article Title: Bradykinin B 2 Receptor Interacts with Integrin ?5?1 to Transactivate Epidermal Growth Factor Receptor in Kidney Cells

doi: 10.1124/mol.110.064840

Figure Lengend Snippet: Western blot analysis of mIMCD-3 lysates. Western blot analyses of mIMCD-3 lysates (40 μg of total protein) were performed with commercially available anti-integrin antibodies to demonstrate the expression of these integrins on a protein level. The bands of α2 (155 kDa), α3 (150 and 130 kDa), α5 (150 kDa), α6 (140 kDa), αV (150 kDa), β1 (130 kDa), and β6 (97 kDa) are indicated.

Article Snippet: Antibodies against integrin α2, α5, α6, β6, and β1 subunits, blocking antibodies against α3, αV, and β3 integrins, MMP-8 siRNA, MMP-13 siRNA, integrin α2 siRNA, integrin α5 siRNA, integrin β1 siRNA, and control siRNA were from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Western Blot, Expressing

Journal: Molecular Pharmacology

Article Title: Bradykinin B 2 Receptor Interacts with Integrin ?5?1 to Transactivate Epidermal Growth Factor Receptor in Kidney Cells

doi: 10.1124/mol.110.064840

Figure Lengend Snippet: Transfection of mIMCD-3 cells with integrin and MMP siRNAs decreases BK-induced ERK activation. mIMCD-3 cells were nucleofected either with 100 nM siRNA for integrin α5β1 alone (-α5β1) or with combinations of MMP-8 siRNA (-α5β1-MMP-8) and/or MMP-13 siRNA (-α5β1-MMP-13), or with combinations of all siRNAs (-α5β1- MMP-8-MMP-13), or with the same amount of control siRNA (control). Forty-eight hours after nucleofection, cells were stimulated with vehicle or 100 nM BK (A) or with 1 ng/ml EGF (B) for 5 min, lysed, and analyzed for ERK phosphorylation. ERK phosphorylation was measured as described under Materials and Methods. Bars represent intensities of phospho-ERK bands relative to total ERK expressed as fold of basal (cells treated with vehicle). Experiments were performed three times in duplicate. Data are presented as mean + S.E.M. **, p < 0.01 compared with control BK-treated cells. ANOVA -α5β1 compared with -α5β1-MMP-8, -α5β1-MMP-13, or with -α5β1-MMP-8-MMP-13, not significant.

Article Snippet: Antibodies against integrin α2, α5, α6, β6, and β1 subunits, blocking antibodies against α3, αV, and β3 integrins, MMP-8 siRNA, MMP-13 siRNA, integrin α2 siRNA, integrin α5 siRNA, integrin β1 siRNA, and control siRNA were from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Transfection, Activation Assay

Journal: Molecular Pharmacology

Article Title: Bradykinin B 2 Receptor Interacts with Integrin ?5?1 to Transactivate Epidermal Growth Factor Receptor in Kidney Cells

doi: 10.1124/mol.110.064840

Figure Lengend Snippet: Transfection of mIMCD-3 cells with integrin α5β1 and MMP siRNAs decreases BK-induced EGFR phosphorylation. Cells were nucleofected with 100 nM α5β1 siRNA (-α5β1) or -α5β1 with a combination of either MMP-8 siRNA (-α5β1-MMP-8) or MMP-13 siRNA (-α5β1-MMP-13); with a combination of all siRNAs (-α5β1- MMP-8-MMP-13); or with the same amount of control siRNA (control), as described under Materials and Methods. Forty-eight hours after nucleofection, cells were stimulated with vehicle or 100 nM BK (A) or with 1 ng/ml EGF (B) for 5 min, lysed, and analyzed for EGFR phosphorylation as described under Materials and Methods. Experiments were performed at least three times. Data are presented as mean + S.E.M. **, p < 0.01 compared with control BK-treated cells. ANOVA (-α5β1) compared with -α5β1-MMP-8, -α5β1-MMP-13, or -α5β1- MMP-8-MMP-13, not significant. C, Western blot analyses of lysates of mIMCD-3 cells transfected with either scrambled siRNA or siRNAs for α5, β1, MMP-8, and MMP-13 (40 μg of total protein) were performed with commercially available antibodies against α5 and β1 integrin subunits, MMP-8 and MMP-13 to demonstrate down-regulation of these proteins. Blots were stripped and re-probed with antibody against GAPDH to control for the specificity of silencing and protein loading.

Article Snippet: Antibodies against integrin α2, α5, α6, β6, and β1 subunits, blocking antibodies against α3, αV, and β3 integrins, MMP-8 siRNA, MMP-13 siRNA, integrin α2 siRNA, integrin α5 siRNA, integrin β1 siRNA, and control siRNA were from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Transfection, Western Blot

Journal: Molecular Pharmacology

Article Title: Bradykinin B 2 Receptor Interacts with Integrin ?5?1 to Transactivate Epidermal Growth Factor Receptor in Kidney Cells

doi: 10.1124/mol.110.064840

Figure Lengend Snippet: BK induces complex formation between EGFR and α5β1 integrin. Lysates from mIMCD-3 cells treated with vehicle, 100 nM BK, or 1 ng/ml EGF were immunoprecipitated with anti-α5β1 integrin antibody as described under Materials and Methods. Immunoblotting was performed with antibodies against EGFR (A) and BK B2 receptor (B). The blots shown are representative of four experiments. A, coimmunoprecipitation experiments show that α5β1 integrin and EGFR coimmunoprecipitate and that their association can be increased by stimulation of mIMCD-3 cells with 100 nM BK but not with EGF. Inset, representative Western blot with antibody against EGFR showing immunoreactive band at 175 kDa. Blot was stripped and re-probed with antibody against α5 integrin to control for immunoprecipitation and protein loading. Immunoreactive band at 150 kDa is shown. B, BK B2 receptor coimmunoprecipitates with α5β1 integrin. Inset, representative Western blot with antibody against BK B2 receptor showing immunoreactive duplet at 42/40 kDa. Blot was stripped and reprobed with antibody against α5 integrin to control for immunoprecipitation and protein loading. Immunoreactive band at 150 kDa is shown. IP, immunoprecipitation; IB, immunoblot.

Article Snippet: Antibodies against integrin α2, α5, α6, β6, and β1 subunits, blocking antibodies against α3, αV, and β3 integrins, MMP-8 siRNA, MMP-13 siRNA, integrin α2 siRNA, integrin α5 siRNA, integrin β1 siRNA, and control siRNA were from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Immunoprecipitation, Western Blot