Association of cingulin with α-tubulin. (A) Coimmunoprecipitation of cingulin with α-tubulin. HA-cingulin (HA-CGN) or HA (HA) was exogenously overexpressed in HEK293 cells (Exo, exogenous), and their extracts were pulled down with an anti–α-tubulin antibody (α-Tub Ab). Black lines indicate that intervening lanes have been spliced out. IP, immunoprecipitation; WB, Western blotting. (B) Cingulin domain analysis for its association with α-tubulin. α-Tubulin binds to the head domain of cingulin. FL, full length. (C) Coimmunoprecipitation of endogenous cingulin with α-tubulin. Eph4 extracts were pulled down with anti-cingulin or anti–α-tubulin antibody. (D) Generation of cingulin knockdown (KD) Eph4 cells. (E) Immunofluorescence for α-tubulin in wild type, cingulin KD cells, and KD cells expressing an exogenous RNAi-resistant cingulin sequence (cingulin revertant [CGN] Rev.). Bar, 5 µm. The relative signal intensity of immunofluorescence was quantified for α-tubulin (top line) and ZO-1 (bottom line) for 10 cells.