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. 2017 Feb 23;8(2):e2627. doi: 10.1038/cddis.2017.51

Figure 1.

Figure 1

ULK2 binds to and phosphorylates CARMA2sh. (a) HEK293T cells were cotransfected with a plasmid encoding for HA-tagged CARMA2sh together with a FLAG-tagged expression vector empty or encoding for ULK2. About 24 h later, lysates were immunoprecipitated with anti-FLAG or anti-myc control antibodies and analyzed for coprecipitating HA-CARMAsh by western blot assay. (b) HaCaT cells were transfected with either an empty plasmid or encoding for HA-tagged CARMA2sh. About 24 h later, cells were lysed and immunoprecipitated with anti-HA or anti-myc control antibodies, and analyzed for coprecipitating endogenous ULK2 protein by western blot assay. (c) HEK293T cells were cotransfected with a plasmid encoding for HA-tagged CARMA2sh together with an expression vector encoding for ULK2. About 24 h later, CARMA2sh expression was analyzed by immunoblot assay probed with anti-HA. Where indicated, the cell lysate was treated with CIP (0.5 U/μg lysate) for 30 min at 37 °C and/or a mixture of phosphatase inhibitors (i-CIP). (d) Lysates from HEK293T separately transfected with the indicated expression plasmids were immunoprecipitated and tested in a mixed beads in vitro kinase assay as described in Material and Methods section. The slower migration in SDS/PAGE of wt ULK2 compared to ULK2K39I is due to ULK2 autophosphorylation (data not shown)