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. 2019 Mar 15;11(3):1521–1530.

Figure 2.

Figure 2

S48 phosphorylation by ERK is required for KLF8 to interact with NEDD4. A. The Serine 48 residue of KLF8 is responsible for the binding of NEDD4 and KLF8. HA-KLF8, wild-type or mutants indicated, was co-expressed with Myc-NEDD4 in HEK293 cells for 48 hours. Co-IP and IB were carried out similarly as described in Figure 1. B. ERK activity is required for the binding of KLF8 to NEDD4. Similarly treated HEK293 cells overexpressing Myc-NEDD4 and HA-KLF8 were treated with the MEK inhibitor PD98059 (50 mM) or DMSO for 30 min prior to cell lysate preparation for the similar co-IP assay. Effective inhibition of ERK activity was verified by IB, the WCL with an antibody against active ERK (p-Erk) and an antibody against total ERK (Erk). Expression of the KLF8 and NEDD4 was confirmed by immunoblotting the WCL with b-actin serving as a loading control.