Fig. 4.
p57Kip2 enhances DA cell differentiation in vitro by direct interaction with the amino-terminal domain of Nurr1. (A) HEK 293 cells were transfected with expression vectors encoding Nurr1, Nurr194–598, Nurr1183–598, or Nurr11–355 either alone or together with a HA-p57Kip2 expression vector. Nuclear cell extracts were immunoprecipitated by using anti-p57Kip2 antibodies, and immune complexes were subjected to immunoblotting by using anti-HA (Upper) or anti-Nurr1 (Lower) antibodies. (B) p57Kip2 is unable to cooperate with interaction-deficient amino-terminally truncated Nurr1 derivatives. MN9D cells were cotransfected with expression vectors encoding EGFP and Nurr1, Nurr194–598, Nurr1183–598, or Nurr11–355 alone or together with p57Kip2 expression vector. The number of differentiated cells was counted 3 days after transfection as in Fig. 4, and the fold increase was calculated. Error bars represent standard deviation. p57Kip2 is required for Nurr1-induced differentiation but not cell cycle arrest. (C) MN9D cells were transfected with expression vectors encoding EGFP and Nurr1 either alone or together with p57Kip2 or antisense p57Kip2 (asp57) expression vectors. The number of differentiated EGFP-expressing cells was counted 3 days after transfection as in Fig. 4, and the fold increase was calculated. Error bars represent standard deviation. Average values of quadruplicates are shown. (D) MN9D cells were transfected with expression vectors encoding EGFP and Nurr1 or Nurr11–355 either alone or together with p57Kip2 or p57 CKmut expression vectors.