Role of AMPK-mediated phosphorylation of cingulin in its association with MTs. (A) AMPK target motifs in cingulin sequences (yellow shadowing). (B) Coimmunoprecipitation of HA-cingulin with V5-AMPKα1. Binding occurs between cingulin and AMPKα1 (yellow arrowhead, V5-AMPKα1). Black lines indicate that intervening lanes have been spliced out. WB, Western blot. (C) Phosphorylation level of wild-type and dephosphomimetic mutants of cingulin. As to the relative intensity, the ratio of intensity of Pro-Q staining to Coomassie brilliant blue (CBB) staining in wild type (WT) was normalized to 1.0, and the results are expressed as means ± SE (error bars; n = 3). (D) SIM images of the immunofluorescence in Eph4 cells treated with the AMPK inhibitor compound C. Bar, 5 µm. The α-tubulin association with TJs was disturbed by the AMPK inhibitor compound C. The relative signal intensity of immunofluorescence was quantified for α-tubulin (top line) and cingulin (bottom line) for 10 cells. CGN, cingulin; α-Tub, α-tubulin.