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Fig. 4. DLC1 interaction with 14-3-3 proteins requires phosphorylation of Ser327 and Ser431. HEK293T cells were transiently transfected with GFP-tagged DLC1 WT, S327A, S431A, or S327/431A expression vectors. Whole-cell extracts were subjected to a pull-down with GST-14-3-3 ${tau}$ beads and bound proteins were separated by SDS-PAGE. DLC1 variants were detected with a GFP-specific antibody (top panel). The integrity of recombinant GST-14-3-3 ${tau}$ was verified by probing the membrane with GST-specific antibody (middle panel), and expression of DLC1 variants was verified by immunoblotting of whole-cell extracts with GFP-specific antibody (bottom panel). (B) An HA-tagged 14-3-3 ${tau}$ expression vector was transiently transfected into HEK293T cells along with DLC1 WT, S327/431A or empty vector (con). DLC1 variants were immunoprecipitated from whole-cell extracts with GFP-specific antibody and immune complexes were separated by SDS-PAGE. Coprecipitated 14-3-3 ${tau}$ was detected by western blotting with HA-specific antibody (middle panel). Immunoprecipitation of DLC1 variants was verified by probing the membrane with GFP-specific antibody (top panel). Equal expression of 14-3-3 ${tau}$ was verified by immunoblotting of whole-cell extracts with HA-specific antibody (bottom panel). (C) HEK293T cells were transiently transfected with expression plasmids encoding Flag-tagged DLC1 WT or S327/431 and empty vector (–) or GFP-tagged PKD1 (+), respectively. Whole-cell extracts were subjected to GST-14-3-3 ${tau}$ pull-downs and analyzed as described in A. Expression of PKD1 and DLC1 variants was verified by immunoblotting of whole-cell extracts with GFP- and Flag-specific antibodies, respectively (bottom panels). (D) HEK293T transiently expressing GFP-DLC1 WT or S327/431A were treated left untreated or treated with 100 nM okadaic acid for 2 hours or 1 µM PDBu for 15 minutes. Whole-cell extracts were subjected to GST-14-3-3 ${tau}$ pull-downs and analyzed as described in A. (E) HEK293T cells transiently expressing GFP-tagged DLC1 WT, S327/431A or S327/431D were left untreated (–) or treated with 1 µM PDBu (+) for 15 minutes. Whole-cell extracts were subjected to GST-14-3-3 ${tau}$ pull-downs and analyzed as described in A. (F) HEK293T cells transiently expressing GFP-tagged DLC1 WT, S327/431A, S327/419/431A, or S236/327/419/431A were left untreated (–) or treated with 1 µM PDBu (+) for 15 minutes. Whole-cell extracts were subjected to GST-14-3-3 ${tau}$ pull-downs and analyzed as described in A. (G) Recombinant GST-DLC1(aa242-569) proteins were incubated in kinase buffer containing [ ${gamma}$ -³²P]ATP in the absence (left lanes) or presence of purified Myc-tagged PKD1 (right lanes). Proteins were separated by SDS-PAGE and transferred to membrane. Incorporation of radioactive phosphate was analyzed using a PhosphoImager (top panel), followed by immunoblotting with GST- and Myc-specific antibodies to verify equal loading (bottom panels). The GST-DLC1 fusion proteins and Myc-PKD1 are indicated with arrows.

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