Figure 5. Deregulated Torso activity at the posterior pole inhibits PGC formation.

(A) HEK293T cells were transfected with FLAG-tagged GCL^WT, HA-tagged Torso (either WT or Deg mutant), and MYC-tagged dominant negative CUL3 (DN CUL3), as indicated. Cell lysates were immunoprecipitated (IP) with anti-FLAG resin, and immunocomplexes were probed with the indicated antibodies. For Torso^Deg amino acids IENIG were mutated to alanines.

(B) HEK293T cells were transfected with MYC-tagged wild-type Ubiquitin (Ub), either HA-tagged Torso or HA-tagged Torso^Deg, and either FLAG-tagged GCL^WT or FLAG-tagged GCL^E100K. Twenty-four hours post-transfection, cells were treated with MG132 for 3 hours before collection for immunoprecipitation (IP) under denaturing condition and immunoblotted as indicated. The bracket on the right indicates a ladder of bands with a relative molecular mass of >150 kDa which, presumably, are poly-ubiquitylated species of Torso. WCE, whole-cell extract.

(C) Embryos from torso^+/− (torso^+/HH), torso^−/− (torso^HH/WK), or torso^Deg/− (torso^Deg/HH) females were immunostained with antibodies recognizing Vasa (green) to assess PGC formation and dpERK (magenta) to assess Torso signaling activity. Presence of the rounded nuclei (Blue) around the periphery of the embryos was used to select and assess embryos of nuclear cycle 12–13. Arrowhead points at the absence of PGC at posterior pole. Scale bar = 50 μm.

See also Figure S6.