Figure 5.

SOCS3 interacts with MT plus-end-binding proteins. A, ECs with ectopic expression of Myc-SOCS3 were treated with HKSA, with or without low-dose nocodazole (low ND, 0.05 nM); EC permeability was monitored by TER; n = 5. B, interaction of Myc-SOCS3 with MT-associated proteins analyzed by co-IP assay with CLIP-170 and CLASP2 antibodies and immunoblotting for Myc. EB1 was monitored as a known interacting partner of CLIP and CLASP. SOCS3 and EB1 contents in total cell lysates were monitored to verify equal protein amounts used for IP. Shown are representative results of four independent experiments. C, ECs cotransfected with GFP-tagged CLIP-170, CLASP2, EB1, or β-tubulin and Myc-SOCS3 were used for co-IP assays with GFP antibody and western blot detection of Myc-SOCS3. Myc-SOCS3 detection in total cell lysates was used to verify equal inputs. Shown are representative results of five independent experiments. D, co-IP of EC coexpressing GFP-tagged CLIP-170, CLASP2, or EB1 and Myc-SOCS3 using Myc antibody for co-IP followed by probing for GFP. Total cell lysates probed for Myc and GFP served as normalization controls. Shown are representative results of four independent experiments; TF: transfection.