Rab18 directly interacts with KNT1. (a) Representative image of a cell transfected with GFP-Rab18 WT and RFP-vinculin and immunostained with an antibody against KNT1. The insets show regions (white squares) where GFP-Rab18 and KNT1 colocalize in proximity of the plasma membrane with the FA protein vinculin (cyan). Scale bar: 10 µm; insets: 1 µm. (b) U2OS cells were transiently transfected with GFP, GFP-Rab18 WT, GFP-Rab18 Q67L, or GFP-Rab18 S22N, lysed, and subjected to IP with GFP or control magnetic agarose beads. Whole-cell lysates (WCL) and immunoprecipitates (IP) were subjected to Western blot analysis using the indicated antibodies. (c) Schematic representation of KNT1 FL and deletion mutants used in this study. The green square indicates the ER membrane-binding domain of KNT1. (d) U2OS cells were transiently cotransfected with HA-Rab18 and GFP, GFP-KNT1 FL, TM, K1, KNT⁺, or KNT⁻, lysed, and immunoprecipitated with GFP magnetic agarose beads. WCL and IP were subjected to Western blot analysis using the indicated antibodies. (e) U2OS cells were transiently cotransfected with HA-Rab18 and GFP, GFP-KNT1 FL, or Δ201–445, lysed, and immunoprecipitated with GFP magnetic agarose beads. WCL and IP were subjected to Western blot analysis using the indicated antibodies. (f) Top: Coomassie blue staining of bacterially expressed and purified GST, GST-KNT1 (201–445), and His-Rab18 Q67L. Bottom: Purified GST or GST-KNT1 (201–445) was incubated with purified His-Rab18 Q67L. Samples were subjected to affinity chromatography followed by Western blot analysis using antibodies specific to GST and Rab18.