MARK family kinase activity is necessary for haptotaxis. (A) Several LKB1 substrates are not required for haptotaxis. Substrates not expressed were not tested in haptotaxis experiments and are listed as not available (N/A). (B) MARK family kinases are required for haptotaxis in LKB498 cells. (addback, n = 125; addback + MARK2, 3, and 4 knockdown [KD], n = 106.) (C) Western blots showing MARK inhibitor (MARKinh) 39621 (20 µM) validation in LKB498 cells. Add, addback; IP, immunoprecipitation; IB, immunoblot. (D) MARK inhibitor blocks haptotaxis in LKB498 cells. (addback, n = 133; MARK inhibitor, n = 92.) (E) Western blots validating effect of dominant-negative (DN) MARK3 overexpression in LKB498 cells. (F and G) DN-MARK3 blocks haptotaxis of LKB498 cells in 2D (F) and 3D (G). (2D: addback, n = 130; DN-MARK3, n = 131. 3D: addback, n = 48; DN-MARK3, n = 40.) All data are shown as mean ± 95% confidence intervals. *, P < 0.05; **, P < 0.01; ***, P < 0.001 by two-tailed unpaired t test. v, velocity; D/T, displacement (D) over the total path length (T).