Fig. 8: Tpx2-like motifs and Serine 112 phosphorylation are essential for Bora function in mitotic entry in Xenopus egg extracts.

a Schematic for the structure–function analysis of Bora in mitotic entry in Xenopus egg extracts. Interphase extracts (red) were depleted with Bora antibodies and 30 min later supplemented with Bora fragments (pre-phosphorylated by the ERK kinase) and with the recombinant Gwl^K72M hyperactive kinase to force mitotic entry. Samples were collected at different time-points and analyzed by Western blot (0, 20, 40, 60 min) using mitotic markers to determine whether the extracts enter into mitosis (green) or not. The Western blot shows endogenous Bora levels before (Input) and after (Sn: supernatant) immunodepletion (Ip) from Xenopus egg extracts. b Interphase extracts were immunodepleted using anti-Bora antibodies and 30 min later supplemented with a recombinant hyperactive Gwl^K72M and Bora^1–224 (lanes 2–5), Bora^35–157 (lanes 6–9), Bora^1–224 (lanes 11–14), Bora^{18–157 [S27A T52A]} (lanes 15–18), Bora^{18–120 [S27A T52A]} (lanes 19–22). A fraction of the extracts was collected at the indicated time-points (0, 20, 40, 60 min) and analyzed by Western blot using specific antibodies to monitor the levels of huGwl, ERK, Plk1, huBora and phosphorylation of Plx1 on T201 (pPlk1), PP1 phosphatase on threonine 320 (pPP1), Cdk on Tyr15 (pTyr), and ERK on Thr202 and Tyr204. The green and red areas at bottom represent high and low CyclinB-Cdk1 (MPF) activity periods. c Interphase extracts were immunodepleted using anti-Bora antibodies and 30 min later supplemented with recombinant hyperactive Gwl (Gwl^K72M) and Bora^{18-120 [S27A T52A]} (lanes 2–5), Bora^{18–120 [S27A T52A F25D Y31D]} (lanes 6–9), Bora^{18-120 [S27A T52A F103D F104D]} (lanes 10–13). A fraction of the extracts was collected at the indicated time-points (0, 20, 40, 60 min) and analyzed by Western blot using specific antibodies to monitor the levels of Gwl, ERK, Plk1, and Bora as well as phosphorylation of Plx1 on T201 (pPlk1), PP1 phosphatase on threonine 320 (pPP1), and Cdk on Tyr15 (pTyr). The green and red areas at bottom represent high and low CyclinB-Cdk1 (MPF) activity periods.