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. 2016 May 19;7(5):e2230. doi: 10.1038/cddis.2016.109

Figure 2.

Figure 2

praja2 ubiquitylates KSR1. (a) HEK293 cells were transfected with HA-ubiquitin and flag-praja2 or flag-praja2RM. Twenty-four hours after transfection, cells were treated with MG132 (20 μM) for 8 h. Lysates were subjected to immunoprecipitations with anti-KSR1 and immunoblotted with anti-HA and anti-flag antibodies. (b) Cells were transfected with HA-ubiquitin, serum-deprived overnight and stimulated with EGF (100 ng/ml) for the indicated time points. Lysates were subjected to immunoprecipitations with anti-KSR1 and immunoblotted with anti-HA and anti-KSR1 antibodies. (c) Cells were transfected with HA-ubiquitin and flag-praja2 or flag-praja2RM and processed as in panel (b). (d) HEK293 cells were transfected with HA-ubiquitin and control (siRNAc) or SMARTpool siRNApraja2. Twenty-four hours after transfection, cells were either left untreated or stimulated with isoproterenol in the presence of MG132. Lysates were subjected to immunoprecipitations with anti-KSR1 and immunoblotted with anti-HA and anti-KSR1 antibodies. (e) Same as in panel (d), with the exception that the cells were stimulated with Fsk (40 μM, 30 min). (f) In vitro-translated, 35S-labeled KSR1 was incubated with anti-flag precipitates (flag-praja2 or flag-praja2RM) isolated from growing cells and his6-tagged ubiquitin, in the presence of E1 and UbcH5c (E2). The reaction mix was denatured, size-fractionated on SDS-PAGE and analyzed by autoradiography. A fraction of the reaction mixture was immunoblotted with anti-flag antibody (lower panel)