Figure 2. praja2 ubiquitylates and degrades MOB1.

(a–d) HEK293 cells were transiently co-transfected with CMV or flag-praja2 (either wild-type or RING mutant) and HA-MOB1A (a) or HA-MOB1B (c) vectors. Lysates were immunoblotted with the indicated antibodies. Quantitative analysis (mean±s.e.m) is reported in the panels (b,d), respectively. *P<0.01 versus CMV; ^#P<0.01 versus flag-praja2, Student’s t-test. (e) Immunoblot analysis of endogenous MOB1 on lysates from cells transiently transfected with CMV, flag-praja2 or flag-praja2rm vector. Tubulin was used as loading control. (f) Cells were transiently co-transfected with CMV or flag-praja2 and HA-MOB1A vectors. Lysates were immunoblotted with the indicated antibodies. The cells were treated for 8 h with MG132 (20 μM) before harvesting. (g) Cells were transiently transfected with CMV or flag-praja2 (either wild-type or RING mutant) and treated for 8 h with MG132 (20 μM) before harvesting. Lysates were immunoblotted with the indicated antibodies. (h) Cells transiently transfected with control siRNA or praja2 siRNA were treated with cycloheximide and harvested at the indicated time points after treatment. Lysates were immunoblotted with the indicated antibodies. (i) Cumulative analysis (mean±s.e.m.) of the experiments shown in h. (j) Cells transiently co-transfected with HA-MOB1A and with either the expression vector for praja2 or praja2rm. CMV vector was included as control. Lysates were immunoblotted with the indicated antibodies. (k) Cumulative analysis of the experiments shown in j. The data are expressed as mean±s.e.m. of three independent experiments. (l) Cells were transiently co-transfected with myc-ubiquitin and flag-praja2 or flag-praja2rm. Cells were treated for 8 h with MG132 (20 μM) before harvesting. Lysates were immunoprecipitated with anti-MOB1 antibody. The precipitates and lysates were immunoblotted with anti-MOB1, anti-myc and anti-flag antibodies. (m) Same as in l, except cells were transiently co-transfected with HA-MOB1A and lysates were immunoprecipitated with anti-HA antibody. Precipitates and lysates were immunoblotted with anti-HA, anti-myc and anti-flag antibodies. (n) In vitro-translated, [³⁵S]-labelled MOB1A was incubated with flag-praja2 or flag-praja2rm (immunopurified from transfected cells) and his6-tagged ubiquitin, in the presence of E1 and UbcH5c (E2). The reaction mixture was denatured, size-fractionated on 12% SDS–PAGE, and analysed by autoradiography. A fraction of the reaction mixture was immunoblotted with anti-flag antibody (lower panel).