Figure 3. WNT5A utilizes the PTK7-JNK pathway to promote cells movements. (A) FLAG-PTK7 and FLAG-ROR2 were overexpressed in HEK 293T cells. After 8h starvation, cells were stimulated for 15 min with WNT5A (200 ng/ml). Cell lysates were subjected to western blot with the mentioned antibodies. pP54 and P54 refer to phosphorylated and non-phosphorylated p54 JNK isoform, respectively. pP46 and P46 refer to phosphorylated and non-phosphorylated p46 JNK isoform, respectively. (B) FLAG-tagged PTK7 and ROR2 were co-expressed with a AP-1 luciferase reporter in HEK 293T cells and luciferase activity was measured as detailed in material and methods. Data are representative of three experiments. Significant difference was determined by Student’s t-test (*P<0.05; **P<0.01). (C) Various constructs expressing full length or truncated forms of PTK7 (**) were overexpressed in HEK 293T cells. After 8h starvation, cells were stimulated for 15 min with WNT5A (200 ng/ml). Cell lysates were subjected to western blot with the mentioned antibodies. Endogenous PTK7 is pointed by one asterisk (*). (D) MEFs isolated from wild-type (WT) or genetrapped ptk7 (PTK7 KO) mice were starved for 8h and stimulated for 15 min with WNT5A (200 ng/ml). Cell lysates were subjected to western blot with the mentioned antibodies. (E) MEFs WT or PTK7 KO were seeded in 6 well plates, starved and incubated with DMEM 1% FCS +/- WNT5A (200ng/mL) and submitted to wound healing. The percentage of wound closure was evaluated after 8 hours. Re-expression of PTK7 (right panel: western blot of protein extracts using the mentioned antibodies) was able to partially rescue the loss of cell migration in PTK7 KO cells. Data are representative of three experiments. Significant difference was determined by Student’s t-test (*P<0.05; **P<0.01). (F) JNK activity is required for WNT5A-induced wound healing in MEFs. As in (E) except that MEFs were treated with two JNK inhibitors CAS 129-56-6 (CAS, 100μM) and SP600125 (100μM) (left). On the right, lysates of MEFs were probed with the indicated antibodies.
Image published in: Martinez S et al. (2015)
© 2015 by The American Society for Biochemistry and Molecular Biology, Inc. This image is reproduced with permission of the journal and the copyright holder. This is an open-access article distributed under the terms of the Creative Commons Attribution license
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