Figure 8.
Figure 8. The actin-binding domain and IQ motifs of IQGAP1 must be on the same molecule for IQGAP1 to function. (A) Schematic showing the naming of additional IQGAP1 truncation constructs fused to GFP or mCherry. (B) The tagged constructs shown in A were transduced into control or IQGAP1 shRNA (KD) iHUVECs grown on collagen gels as indicated (“GFP” denotes monolayers expressing GFP alone). After 2 d of reexpression of the truncation constructs (which corresponds to 3 d of transduction of the control/KD constructs), the cells were lysed and their proteins resolved with SDS-PAGE and probed using Western blotting. The representative Western blots shown were probed for IQGAP1, mCherry, and actin using the indicated antibodies. Blots shown are from different gels/transfers of the same samples. Note that mCherry construct IQGAP1Δ1-3 (Δ1-3 in red) is not detected with the IQGAP1 antibody because it lacks the coiled-coil domain that is recognized by the antibody. Arrow denotes the relative mobility of endogenous IQGAP1. (C) Immunofluorescence images of the mCherry and GFP IQGAP1 constructs transduced in iHUVECs as in B. For each channel, the images shown were collected with the same settings and processed identically to preserve relative intensity differences. Arrowheads denote observed junctional localization of the indicated construct. The images shown are representative of more than three independent experiments. Scale bar represents 25 µm. (D) iHUVECs grown on collagen gels were transduced with the indicated constructs and subjected to the standard TEM assay (as described in Fig. 7 and the Materials and methods). The percent TEM is shown for the indicated IQGAP1 KD coexpression combinations along with control samples (black bars); no-knockdown expressing GFP alone and no-knockdown but TEM blocked with the function-blocking anti-PECAM antibody hec7 (α-PECAM). Data shown are the average and standard deviation from three experiments, each of which comprised triplicate samples for each condition. *, P < 0.01 compared with the knockdown-FL rescue control.

The actin-binding domain and IQ motifs of IQGAP1 must be on the same molecule for IQGAP1 to function. (A) Schematic showing the naming of additional IQGAP1 truncation constructs fused to GFP or mCherry. (B) The tagged constructs shown in A were transduced into control or IQGAP1 shRNA (KD) iHUVECs grown on collagen gels as indicated (“GFP” denotes monolayers expressing GFP alone). After 2 d of reexpression of the truncation constructs (which corresponds to 3 d of transduction of the control/KD constructs), the cells were lysed and their proteins resolved with SDS-PAGE and probed using Western blotting. The representative Western blots shown were probed for IQGAP1, mCherry, and actin using the indicated antibodies. Blots shown are from different gels/transfers of the same samples. Note that mCherry construct IQGAP1Δ1-3 (Δ1-3 in red) is not detected with the IQGAP1 antibody because it lacks the coiled-coil domain that is recognized by the antibody. Arrow denotes the relative mobility of endogenous IQGAP1. (C) Immunofluorescence images of the mCherry and GFP IQGAP1 constructs transduced in iHUVECs as in B. For each channel, the images shown were collected with the same settings and processed identically to preserve relative intensity differences. Arrowheads denote observed junctional localization of the indicated construct. The images shown are representative of more than three independent experiments. Scale bar represents 25 µm. (D) iHUVECs grown on collagen gels were transduced with the indicated constructs and subjected to the standard TEM assay (as described in Fig. 7 and the Materials and methods). The percent TEM is shown for the indicated IQGAP1 KD coexpression combinations along with control samples (black bars); no-knockdown expressing GFP alone and no-knockdown but TEM blocked with the function-blocking anti-PECAM antibody hec7 (α-PECAM). Data shown are the average and standard deviation from three experiments, each of which comprised triplicate samples for each condition. *, P < 0.01 compared with the knockdown-FL rescue control.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal