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S48 phosphorylation is required for KLF8 ubiquitylation by NEDD4. Myc-tagged NEDD4 was co-expressed with HA-KLF8 or its mutants S48A and S48D in HEK293 cells for 48 hours followed by 12-hour treatment with MG132. Whole cell lysates were prepared for IP with anti-HA followed by IB with anti-ubiquitin. Interaction between KLF8 and NEDD4 was examined by co-IP similarly. Expression of the KLF8 and NEDD4 was confirmed by immunoblotting the WCL with b-actin serving as a loading control.
