Fig. 3.
Fig. 3. In vitro association of Felic, Lyn, and Cdc42. / (A) HEK293 cells were transfected with cDNA for pCMVFLAG-Felic and pCDNA3.1-Lyn. 24 h after transfection, cells were harvested, and proteins immunoprecipitated (IP) with polyclonal antibody against Lyn were resolved on 10% SDS-PAGE. After being transferred onto PVDF, the membrane was blotted with monoclonal antibody (mAb) against FLAG. Detection was performed using enhanced chemiluminscence. Blotting of whole cell lysates (WCLs) of transfected cells show position of Felic (arrowhead). To confirm specific binding between Lyn and Felic, an isotypic control, rabbit anti-Syk polyclonal antibody, was used and failed to coprecipitate FLAG-tagged Felic from Felic-overexpressing cells. (B) HEK293 cells were transfected with cDNA for Felic and Src. In these experiments, proteins immunoprecipitated with polyclonal antibody against c-Src were resolved by SDS-PAGE, followed by transfer to PVDF and blotting with anti-FLAG mAb. (C) HEK293 cells were transfected with cDNA for Felic and wild-type and mutant forms of Cdc42. V12Cdc42 and Q61L Cdc42 are constitutively activated forms, whereas N17Cdc42 is the inactive form. The cDNAs for Cdc42 forms encoded the HA tag. At 24 hours after transfection, cells were harvested and proteins immmunoprecipitated with mAb against HA were resolved by 10% SDS-PAGE, transferred onto PVDF filter, and blotted with mAb against FLAG. Lane 2 represents WCLs of Felic-transfected cells. Lane 9 represents a duplicate of experimental conditions shown in lane 6. Lanes 6, 8, and 9 show association of Felic with activated Cdc42. Lanes 5 and 8 show the absence of association of Felic with inactive or wild-type forms of Cdc42. (D) HEK293 cells were transfected with cDNA for pCMVFLAG-Felic and pCDNA3.1-V12Cdc42HA and varying amounts of pcDNA3.1-Lyn (0-8 μg). Plasmid pcDNA3 was added to keep the total cDNA transfected equivalent at 25 μg between transfection conditions. Proteins were immunoprecipitated with mAb against HA, and blotting was performed with FLAG mAb. Equivalent amount of Felic was expressed as demonstrated by blotting.

In vitro association of Felic, Lyn, and Cdc42.

(A) HEK293 cells were transfected with cDNA for pCMVFLAG-Felic and pCDNA3.1-Lyn. 24 h after transfection, cells were harvested, and proteins immunoprecipitated (IP) with polyclonal antibody against Lyn were resolved on 10% SDS-PAGE. After being transferred onto PVDF, the membrane was blotted with monoclonal antibody (mAb) against FLAG. Detection was performed using enhanced chemiluminscence. Blotting of whole cell lysates (WCLs) of transfected cells show position of Felic (arrowhead). To confirm specific binding between Lyn and Felic, an isotypic control, rabbit anti-Syk polyclonal antibody, was used and failed to coprecipitate FLAG-tagged Felic from Felic-overexpressing cells. (B) HEK293 cells were transfected with cDNA for Felic and Src. In these experiments, proteins immunoprecipitated with polyclonal antibody against c-Src were resolved by SDS-PAGE, followed by transfer to PVDF and blotting with anti-FLAG mAb. (C) HEK293 cells were transfected with cDNA for Felic and wild-type and mutant forms of Cdc42. V12Cdc42 and Q61L Cdc42 are constitutively activated forms, whereas N17Cdc42 is the inactive form. The cDNAs for Cdc42 forms encoded the HA tag. At 24 hours after transfection, cells were harvested and proteins immmunoprecipitated with mAb against HA were resolved by 10% SDS-PAGE, transferred onto PVDF filter, and blotted with mAb against FLAG. Lane 2 represents WCLs of Felic-transfected cells. Lane 9 represents a duplicate of experimental conditions shown in lane 6. Lanes 6, 8, and 9 show association of Felic with activated Cdc42. Lanes 5 and 8 show the absence of association of Felic with inactive or wild-type forms of Cdc42. (D) HEK293 cells were transfected with cDNA for pCMVFLAG-Felic and pCDNA3.1-V12Cdc42HA and varying amounts of pcDNA3.1-Lyn (0-8 μg). Plasmid pcDNA3 was added to keep the total cDNA transfected equivalent at 25 μg between transfection conditions. Proteins were immunoprecipitated with mAb against HA, and blotting was performed with FLAG mAb. Equivalent amount of Felic was expressed as demonstrated by blotting.

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