Figure 8.

SOCS3/CLIP-170/CLASP2 complex is targeted by cytoskeletal scaffold protein IQGAP1. A, pull-down (PD) assay of Myc-SOCS3 using agarose beads with immobilized full-length IQGAP1 and its ΔC mutant. Bound SOCS3 fraction was detected by immunoblotting with Myc antibody. Probing of total lysates for Myc-SOCS3 was used as a normalization control. B, pull-down of SOCS3 on agarose beads with immobilized IQGAP1 from Myc-SOCS3-expressing EC with siRNA-depleted CLASP2 or CLIP-170. Probing of total lysates for Myc-SOCS3 was used as a normalization control. Bar graphs depict the results of quantitative densitometry of western blot data; n = 5, ∗p < 0.05. C, Myc-SOCS3-expressing ECs with siRNA-depleted IQGAP1 or cells treated with nonspecific (NS) RNA were tested in co-IP assays with CLASP2 or CLIP-170 antibodies. Presence of SOCS3 in immunocomplexes was monitored by western blot with Myc antibody. IQGAP1 depletion was confirmed by western blot. D, ECs were transfected with nonspecific or IQGAP1-specific siRNA, and ectopic expression of Myc-SOCS3 was performed. VCAM1 expression after HKSA challenge EC was determined by western blot. Control probing for β-tubulin and Myc-tag served as normalization and SOCS1 ectopic expression controls, respectively. Bar graphs depict the results of quantitative densitometry of western blot data; n = 4, ∗p < 0.05.