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Fig. 5. KGB-1 can phosphorylate GLH-1; proteosomal or JNK inhibitors increase
GLH-1. (A) In vitro kinase reactions were carried out with
GLH-1-GST (first row) and c-Jun-GST (second row). Inputs of KGB-1 (third row)
and MEK-1 (fourth row), both with HA tags, are shown. In lane 1, basal levels
of GLH-1 or c-Jun phosphorylation occur when incubated with KGB-1 alone. Lane
2 shows phosphorylation levels of GLH-1 and c-Jun when KGB-1 is activated by
MEK-1. A mutated inactive form of KGB-1 was used in lane 3. (B)
HighFive cells were infected with GLH-1-6-His and KGB-1-GST (lanes 5 and 6),
GLH-1-6-His and GST alone (lanes 1 and 2), GLH-1-6-His and CSN-5-GST (lanes 3
and 4) or 6-His-tagged GLH-1 alone (lanes 7 and 8) and grown for 18 hours.
They were then treated (or not) with 1 µM MG132 and grown an additional 5
hours before lysing. GLH-1 levels were measured with anti-His antibody. A
ß-tubulin loading control is shown. (C) Young N2 adult C.
elegans were grown in liquid culture for 6 hours, with 1 µM M6132
added for 0-3 hours; GLH-1 levels were assayed by western blot with an
-tubulin loading control. (D) Under the same conditions as in C,
50 µM SP600125 was added and GLH-1 tested by western blot, again with an
-tubulin control.
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