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. 2018 Mar 5;217(3):1015–1032. doi: 10.1083/jcb.201606055

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© 2018 Kovačević et al.

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Figure 6. — Cullin-3 is required for degradation of RhoB in ECs. (A) HUVECs were transduced with control, Cullin-1, Cullin-2, Cullin-3, or RBX1 shRNA, and cells were fixed and stained at 72 h for RhoB (magenta) and F-actin (red). Bars, 15 µm. (B) Cells were treated as in A, and lysates were analyzed for RhoB, Cullin1, Cullin-2, Cullin-3, and Rac1. (C) HUVECs were treated as in A and seeded in ECIS eight-well arrays at 72 h after infection, and resistance was measured at 4,000 Hz. (n = 3). (D) Quantification of the measured resistance from C at 20 h after seeding is shown. (E) RhoB was immunoprecipitated from lysates of HUVECs, and rabbit IgG antibody was used as a negative control. Input equals 2.5% of the lysate. Samples were analyzed for Cullin-3 and RhoB. (F) HEK293T cells were cotransfected with HA-tagged Cullin-3 and mCherry (control) or mCherry-wtRhoB, -T19N RhoB, or -G14V RhoB. 24 h after transfection, HA–Cullin-3 was immunoprecipitated using anti-HA agarose. Input equals 5% of the lysate. Samples were analyzed for RhoB and HA and GAPDH was used as loading control. Error bars represent SD.