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. 2014 Apr 11;289(21):15080–15093. doi: 10.1074/jbc.M114.563742

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© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

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FIGURE 7. — Experiments demonstrating effects of the LMTK2 kinase on endocytosis and the steady-state plasma membrane abundance of CFTR in CFBE41o- cells. CFBE41o- cells stably expressing WT-CFTR were transfected with the WT-LMTK2Δ-FLAG (WT), KM-LMTK2Δ-FLAG (KM), or vector control (V) and cultured for 48 h on collagen-coated tissue culture plates to form monolayers. Immunoblots (A) and summary of data (B) demonstrating that CFTR endocytosis was linear up to 7.5 min under the cell culture conditions. The plasma membrane (PM) and whole cell lysate (WCL) CFTR was detected with antibody CFF596. CFTR endocytosis was calculated as described in “Materials and Methods” and the Fig. 4 legend. Based on the above results, we examined the effects of the WT-LMTK2Δ-FLAG and KM-LMTK2Δ-FLAG at the 7.5-min time representing the linear portion of CFTR endocytosis. Immunoblots (C, IB: CFTR) and summary of results (D) demonstrating that the KM-LMTK2Δ-FLAG decreased CFTR endocytosis when compared with the WT-LMTK2Δ-FLAG or vector control. E, summary of data demonstrating that the KM-LMTK2Δ-FLAG increased CFTR abundance at the plasma membrane at steady-state when compared with the WT-LMTK2Δ-FLAG or vector control (i.e. endogenous LMTK2). The steady-state plasma membrane CFTR was calculated from the amount of biotinylated CFTR at time zero during the endocytic assays (before warming to 37 °C and without GSH treatment). Immunoblots (C, IB: FLAG) and summary of data (F) examining the plasma membrane abundance and endocytosis of the WT-LMTK2Δ-FLAG and KM-LMTK2Δ-FLAG fragments. Compared with the WT-LMTK2Δ-FLAG, the KM-LMTK2Δ-FLAG fragment was more abundant at the plasma membrane due to decreased endocytosis. Endocytosis of the FLAG-tagged fragments was calculated as described for CFTR. The WCL expression of WT-LMTK2Δ-FLAG or KM-LMTK2Δ-FLAG was used, when appropriate as a loading control to account for small differences in the expression levels of the transiently transfected protein fragments. *, p < 0.05 versus vector control (V). **, p < 0.05 versus WT-LMTK2Δ-FLAG. 3–4 experiments/group. Error bars, S.E.