Detection of IPP complex proteins in αIIbα6Bβ3-active parental cells.

<p>Cell lysates obtained from αIIbα6Bβ3-active parental cells were immunoprecipitated with Abs against PINCH (A), α-parvin (B, C), and ILK (D). The co-precipitates were detected by Abs for α-parvin (A), ILK (B), and PINCH (C, D). IgG means immunoprecipitation (IP) using non-immune control IgG. IB stands for immunoblotting. Arrows indicate the predicted sizes of the indicated proteins. Arrowheads (D) indicate the antibody heavy chains used in the IP. Different mobilities between those of the two IgG antibodies are probably caused by differences in the amino acid compositions of them. </p

Characterization of ILK-deficient mutant cells expressing inactive αIIbα6Bβ3.

<p>(A) Immunoblotting for ILK, PINCH, α-parvin, talin, and kindlin-2. Cell lysates obtained from parental cells with constitutively active αIIbα6Bβ3, ILK-deficient mutant cells with inactive αIIbα6Bβ3, and mutant cells transiently transfected with rat ILK cDNA were electrophoresed on SDS-PAGE gels and immunoblotted with indicated Abs. GAPDH shows an internal loading control. (B) Flow cytometry analysis showing PAC-1 (an activation-specific mAb for αIIbβ3) binding to mutant cells transiently transfected with either ILK plasmid or empty plasmid. Bound PAC-1 was detected with a PE-conjugated secondary mAb. </p

Effects of ILK mutants with defects in either PINCH or parvin binding.

<p>The activation indexes of transfected cells (A, C, E). ILK-deficient mutant cells were transiently transfected with GFP cDNA, GFP-fused wild-type ILK (GFPILK-WT) cDNA, GFP-fused ILK mutant with defective PINCH binding (GFPILK-H99D/F109A/W110A) cDNA (A, E), or GFP-fused ILK mutant with defective parvin binding (GFPILK-M402A/K403A) cDNA (C, E). After transfection, the binding of either PAC-1 (A, C) or fibrinogen (E) to the cells was analyzed by flow cytometry. The activation index was determined by the formula shown in Materials and Methods. A value of 100% represents the maximal binding of PAC-1 or fibrinogen to the cells treated with dithiothreitol. Data represent means ± SD of three independent experiments. ** indicates <i>P</i> < 0.01. Immunoblotting showing protein expression of GFP (B, D), GFP-fused wild-type ILK (GFPILK-WT) (B, D), GFP-fused ILK mutant with defective PINCH binding (GFPILK-H99D/F109A/W110A) (B), and GFP-fused ILK mutant with defective parvin binding (GFPILK-M402A/K403A) (D) in ILK-deficient mutant cells. Cell lysates were electrophoresed and immunoblotted with indicated Abs. </p

Knockdown effects of PINCH, parvins, and kindlin-2 in αIIbα6Bβ3-active parental cells.

<p>αIIbα6Bβ3-active parental cells were transiently transfected with PINCH siRNAs (p157 and p755) (A), α-parvin siRNAs (pa503 and pa761) (C), β-parvin siRNAs (pb900 and pb1011) (C), kindlin-2 siRNAs (k770 and k1733) (E), negative control siRNAs, and scrambled siRNAs. Cell lysates were electrophoresed on SDS-PAGE gels, and the separated proteins were immunoblotted with the indicated Abs. GAPDH and β-actin are shown as internal loading controls. The activation indexes of transfected cells (B, D, F) were calculated using the formula shown in Materials and Methods. A value of 100% implies the maximum PAC-1 binding to the cells treated with dithiothreitol (DTT). Data represent means ± standard deviation (SD) of three (B, F) or four (D) independent experiments. ** indicates <i>P</i> < 0.01.</p