Skip to main content
. 2021 Jan 9;296:100239. doi: 10.1074/jbc.RA120.014232

Figure 4.

Figure 4

SOCS3 associates with MT and inflammatory signaling molecules. A, cells were treated with MT depolymerizing (Colch: Colchicine, 0.5 μM, 1 h) and polymerizing (Tax: Taxol, 0.5 μM, 30 min) agents followed by MT fractionation. SOCS3, CLIP-170, CLASP2, acetylated-tubulin (Ac-tub), and α-tubulin protein contents in MT-enriched fractions were determined by western blot. Probing of total cell lysates with α-tubulin antibody was used to ensure equal input. Bar graphs depict analysis of western blotting data after cell stimulations with vehicle (V), colchicine (C) and taxol (T); n = 5, ∗p < 0.05. B, ECs expressing Myc-tagged SOCS3 were used in co-IP assays with gp130 or JAK2 antibodies; incubation with protein G agarose (Ag) alone served as a negative control. SOCS3 in immunoprecipiates was detected by Myc antibody. Probing of total cell lysates with Myc antibody was used to verify equal IP inputs. C, cells overexpressing SOCS3-WT were used for co-IP with Myc antibody; immunocomplexes were probed with α-tubulin, JAK2, and STAT3 antibodies. Probing of total cell lysates with Myc antibody was used to verify equal IP inputs; TF: transfection. D, control and HKSA-stimulated (6 h) EC with Myc-SOCS3 ectopic expression were lysed, and co-IP assays with Myc antibody followed by probing for JAK2, gp130, and STAT3 were performed. Probing of total cell lysates with Myc antibody was used to verify equal inputs. E, association of SOCS3 with MT in EC expressing Myc-SOCS3 was assessed by co-IP with α- or β-tubulin antibodies followed by probing for Myc. F, ECs expressing GFP-β-tubulin or Myc-SOCS3 were stimulated with HKSA or colchicine followed by co-IP with GFP antibody. SOCS3 interaction with tubulin was detected by western blot with Myc antibody; membranes were reprobed for GFP to verify equal pulldown conditions. Equal volumes of total cell lysates were probed with Myc antibody to confirm SOCS3 ectopic expression. Bar graphs in panels (A, D, F) depict the results of quantitative densitometry of western blots; ∗p < 0.05, n = 5 for each experiment. RDU, relative density units.