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Files in this Data Supplement:
Fig. S1. Change of protein tyrosine phosphorylation before and during PDGF stimulation. (A) WCL blots used in Fig. 2C were reprobed with GST or GST fused with SH2 domains of Crk, CrkL, or PI3 kinase. The bound GST or GST fusion proteins were detected by immunoblotting with anti-GST antibody. (B) Graphs represent changes of tyrosine phosphorylation for indicated proteins from Fig. 2C. These values were the averages of two independent experiments. Values in three top graphs were normalized to those for serum starvation. (C) NIH-3T3 cells infected with pMIGR-1 vector-derived retrovirus or ones expressing wt CrkII or CrkII Y221F. These cells were serum-starved, and the cells were harvested without PDGF stimulation or at 7.5 minutes after 18 ng/ml PDGF stimulation. WCL were subjected for IP with antibody against CrkII. WCL and immunoprecipitates were immunoblotted with various antibodies as indicated.
Fig. S2. Characteristics of focal adhesion disassembly after PDGF stimulation. (A) NIH-3T3 cells infected with empty pSUPER vector-derived retrovirus were super-infected with EYFP fused with Nck2, paxillin, or p130Cas. Expression levels of EYFP or EYFP fused proteins in these cells and the cells from Fig. 3 were detected by immunoblotting with anti-EYFP, CrkI/II, and CrkL antibodies. (B) Serum-starved cells were fixed before or 7.5 minutes after stimulation with 18 ng/ml PDGF. Fixed cells were stained with anti-vinculin/AlexaFluor 647, phalloidin/Texas-red, and Hoechst 33342 for vinculin, F-actin, and DNA, respectively. Images are shown with EYFP or EYFP fusion proteins (green), vinculin (red) and F-actin (blue). Scale bar: 20 µm.
Fig. S3. Crk adaptors are involved in Erk1/2 and Rac1 activation but not in Akt activation during PDGF stimulation of NIH-3T3 fibroblasts. (A) CrkI/II/L-knockdown cells were stimulated with the indicated amounts of PDGF, and harvested before and at various time points after stimulation. WCL stimulated with 18 ng/ml PDGF was also subjected to Rac1 GTPase assay. Various proteins and tyrosine phosphorylated proteins in WCL and pull-down fractions were detected by immunoblotting. (B) NIH-3T3 cells were infected with pSUPER vector only or virus expressing shRNA targeting all Crk family genes. Cells were super-infected with MSCV-puro-derived retrovirus carrying EYFP or EYFP fused with various forms of Crk insensitive to CrkI/II/L shRNA. Cells were harvested before and after stimulation with the indicated amounts of PDGF. Indicated proteins and threonine/tyrosine-phosphorylated Erk1/2 in WCL were detected by immunoblotting.
Movie 1. Time-lapse fluorescent confocal microscopy images of cells before and after PDGF stimulation. Cells were triply infected with pSUPER-cerulean vector, MSCV-puro vector expressing mCherry-Actin (red) and MSCV-puro vector expressing EYFP (green).
Movie 2. Time-lapse fluorescent confocal microscopy images of cells before and after PDGF stimulation. Cells were triply infected with pSUPER-cerulean vector carrying shRNA CrkI/II/L, MSCV-puro vector expressing mCherry-Actin (red) and MSCV-puro vector expressing EYFP (green). The expression of cerulean in each cell was confirmed before starting to acquire images.
Movie 3. As for Movie 2, but using MSCV-puro vector expressing EYFP fused with wt CrkII (green) instead of MSCV-puro vector expressing EYFP.
Movie 4. As for Movie 2, but using MSCV-puro vector expressing EYFP fused with CrkL (green) instead of MSCV-puro vector expressing EYFP.
Movie 5. As for Movie 2, but using MSCV-puro vector expressing EYFP fused with CrkI (green) instead of MSCV-puro vector expressing EYFP.
Movie 6. As for Movie 2, but using MSCV-puro vector expressing EYFP fused with CrkII Y221F (green) instead of MSCV-puro vector expressing EYFP.
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