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Fig. 6. Inca interacts with PAK5. (A) Selective medium streaks of
yeast cells expressing Xenopus Inca and PAK5, either alone or
together, as indicated. Inca+PAK5 allows growth on medium lacking leucine,
tryptophan, histidine and adenine. Positive controls include transfection with
vectors containing T antigen and p53 (Clontech). Streaks on -leu/-trp (lower
panel) shown as a control for transfection. (B) Extracts prepared from
HEK293 cells transfected with expression plasmids encoding a PAK5-GFP
fusion or a Myc epitope-tagged Inca separately or together,
immunoprecipitated with anti-Myc, followed by western blot with antibody for
GFP or Myc. Lanes labeled as total are from lysates prior to
immunoprecipitation. PAK5-GFP precipitates with the anti-Myc antibody, but
only when Myc-tagged Inca is present in the extract. (C) Anti-IncaA
peptide antibody specificity. Fertilized eggs were injected with 1 ng of
synthetic mRNA encoding XInca-GFP, and cultured to stage 20.
Detergent (1% NP40)-solubilized protein was extracted with
1,1,2-trichlorotrifluoroethane (Freon) to remove yolk and the equivalent of
two embryos analyzed by SDS-PAGE/western blot. Both anti-GFP and anti-Inca
recognized a single band of the correct molecular weight (
63 kDa).
(D) Extract from untransfected Xenopus A6 cells
immunoprecipitated with preimmune serum or anti-Inca, followed by western blot
using antiserum raised against PAK5 (residues 122-224, a generous gift of N.
Morin). Immunoprecipitation with anti-Inca enriches the PAK5 signal
several-fold compared with preimmune serum, indicating Inca-PAK5 interaction.
(E) Fluorescent images of a CHO cell cotransfected with plasmids
encoding a PAK5-RFP fusion and Xenopus Inca-GFP fusion showing
extensive overlap. (F) Fibrous structures positive for Inca and PAK5
are nocodazole-sensitive. CHO cells transiently transfected with
PAK5-RFP and XInca-GFP were treated for 1 hour with 3 ng/ml
nocodazole (Sigma), then fixed with methanol and photographed using an
inverted fluorescence microscope. The fibers visible without nocodazole
treatment (E) have disappeared.
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