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. 2007 Oct 22;179(2):247–254. doi: 10.1083/jcb.200705175

Figure 1.

Figure 1.

Monoclonal anti–KRIT-1 (15B2) recognizes authentic KRIT-1 protein in endothelial cells. (A) Immunoprecipitation using mAb anti–KRIT-1 (15B2) and immunoblotting with pAb anti–KRIT-1 (Rb6832) yields a band of ∼80 kD in endothelial and CHO cells. An irrelevant antibody, anti-GAPDH, and nonimmune mouse IgG did not immunoprecipitate the KRIT-1 band. Asterisk indicates that the input level of the lane was changed with Photoshop due to high background to show the absence of a band. (B) Antibody reactivity requires the presence of KRIT-1. siRNA against KRIT-1 (siRNA 530) eliminated the KRIT-1 band immunoprecipitated from BAECs and reduced that in HUVECs by 70%. Negative control siRNA and siRNA directed against GAPDH had no effect on KRIT-1 expression. Blots are representative; BAEC, n = 3; HUVEC, n = 2. (C) KRIT-1 depletion using anti–KRIT-1 siRNA 530 has no effect on the expression of junctional proteins. BAECs treated with anti–KRIT-1 siRNA (530) or control siRNA (NC) were Western blotted for junctional proteins. Although KRIT-1 knockdown of >80% was observed by immunoprecipitation and blotting of KRIT (bottom), no effect on the expression of junction proteins was seen. Blots are representative; n = 3. Black lines indicate that intervening lanes have been spliced out.