1. Drosophila 14-3-3/PAR-5 Is an Essential Mediator of PAR-1 Function in Axis Formation 
Richard Benton, Isabel M. Palacios, Daniel St Johnston 
Developmental Cell 
Volume 3, Issue 5, Pages (November 2002)
DOI: /S (02)

2. Figure 1 Identification of 14-3-3 Proteins as PAR-1 Interactors
(A) Schematic of the PAR-1(N1L) isoform (UBA, ubiquitin-associated domain), the two-hybrid screen bait, and results of the screen.
(B) C. elegans PAR-5 and PAR-1 (kinase/UBA domains) interact in a direct two-hybrid test. Yeast strains were grown on plates selecting for expression of the HIS3 reporter.
(C) Upper panel, interaction of in vitro synthesized 35S-Met-labeled PAR-1 with maltose binding protein (MBP): fusion proteins on amylose beads. The left-hand lane was loaded with 25% of the labeled PAR-1 added to each binding assay. Lower panel, after autoradiography, the gel was stained with Coomassie to visualize the MBP fusion proteins.
(D) Schematic of subfragments used to map the binding site.
Developmental Cell 2002 3, DOI: ( /S (02) )

3. Figure 2 PAR-1 Binds to a Novel Interface in 14-3-3 Proteins
(A) Cartoon representation of a bovine ζ dimer (Liu et al., 1995), with the residues equivalent to those affected in ϵ missense alleles highlighted in green.
(B–D) Yeast two-hybrid liquid β-galactosidase assays using ϵ (B), Raf C terminus (amino acids 419–788) (C), and PAR-1 (D) baits, and preys as indicated beneath each graph. E183K, F199Y, and Y214F are mutant versions of the ϵ prey. Activity units (+/− standard deviation) are arbitrary. The F199Y mutation does not completely abolish interaction with PAR-1, as this strain can still grow, albeit very slowly, on plates selecting for the HIS3 reporter (data not shown).
Developmental Cell 2002 3, DOI: ( /S (02) )

4. Figure 3 PAR-1 Can Phosphorylate a 14-3-3 Binding Site
In vitro kinase assay using α-GFP immunoprecipitates from wild-type or GFP:PAR-1-expressing ovarian extracts incubated with MBP fusions of wild-type or mutant Raf C termini. Lower panel, after autoradiography, the gel was stained with Coomassie to verify that each reaction contained equal amounts of substrate.
Developmental Cell 2002 3, DOI: ( /S (02) )

5. Figure 4 14-3-3 Proteins Are Required for Oocyte Determination
(A) Wild-type stage 4 egg chamber stained for DNA (green) and actin (red), containing a germline cyst, comprising 15 nurse cells and a posterior oocyte, surrounded by a monolayer of somatic follicle cells. Anterior is to the left. The oocyte is enriched for cortical actin and contains compacted DNA within the karyosome (arrow), in contrast to the polyploid nurse cells.
(B) Egg chamber from homozygous ϵj2B10 mutant female stained as in (A). Although one of the germline cells with four ring canals (arrowheads) should differentiate as an oocyte (de Cuevas et al., 1997), both have adopted the nurse cell fate.
(C) Wild-type stage 2 egg chamber, stained for MSPS which marks the posterior MTOC.
(D) ϵj2B10 homozygous mutant egg chamber, stained for MSPS, in which this MTOC is absent.
(E and F) Orb staining in wild-type (E) and ϵj2B10/Df(3R)P14
Developmental Cell 2002 3, DOI: ( /S (02) )

6. Figure 5 14-3-3 Proteins Colocalize with PAR-1
(A) Colocalization of ϵ and (A') GFP:PAR-1(N1S) on the early fusome. (A”) Merged images. (B) Colocalization of ϵ with (B') GFP:PAR-1(N1S) at ring canals. (B”) Merged images. The intense staining in the oocyte nucleus appears to be artifactual, as it is observed with Texas Red- but not FITC-conjugated secondary antibodies. (C) ϵ is detected in the oocyte cytoplasm but does not accumulate at the posterior pole like (C') GFP:PAR-1(N1S). (C”) Merged images. (D and E) FRT ϵj2B10 /FRT GFP;leoP1188/+ egg chambers stained for PAR-1 (red). Cysts lacking GFP (marked by white dots in [D]) (i.e., homozygous for ϵj2B10 and heterozygous for leoP1188) display a fully penetrant defect in oocyte determination, but PAR-1 still localizes to the fusome (D) and the ring canals (E). (F) Genotype as in (D), stained for BAZ, whose localization to circles around ring canals is not affected in mutant clones (arrowheads).
Developmental Cell 2002 3, DOI: ( /S (02) )

7. Figure 6 14-3-3 Proteins Are Required for Oocyte A-P Polarization
(A–C) Localization of STAU in a wild-type stage 10 oocyte (A), an oocyte from ϵj2B10/Df(3R)P14 mutant female (B), and a ϵj2B10 germline clone (C).
(D–F) osk mRNA localization in a wild-type oocyte (D) and ϵj2B10 germline clones (E and F).
(G–I) bcd mRNA localization in wild-type (G), ϵj2B10 germline clone (H), and par-1l(2)k06323/par-1W3 (I) oocytes.
(J) Penetrance of the oocyte polarization defects in ϵj2B10 and par-1 genetic combinations (left-hand side) and mutant combinations (right-hand side).
Developmental Cell 2002 3, DOI: ( /S (02) )

8. Figure 7
Is Required for the Correct Organization of the Oocyte MTs
(A and B) Kin:β-gal localization in wild-type (A) and ϵj2B10 mutant (B) oocytes.
(C and D) FITC-α-tubulin staining of wild-type (C) and ϵj2B10 germline clone (D) oocytes.
Developmental Cell 2002 3, DOI: ( /S (02) )