Monoclonal nanobody sequences and purification.

<p>A, Amino acid sequences of three isolated nanobodies are aligned. Two conserved cysteine residues and three complementarity-determining regions (CDRs) are indicated. B, Coomassie-stained gel demonstrating nanobody purification by Ni-chromatography. FT, flow through; E1–3, first to third elution; Ni, the Ni-resin before elution; peri, periplasm extract used for purification; BSA, bovine serum albumin. Asterisks indicate the positions of the purified nanobodies, NbP1 and NbP7.</p

Immunodetection of specific antigens in embryo lysates.

<p>For western blot analysis, lysates of midgastrula (st. 10.5) and early neurula (st. 14) embryos were separated on SDS-PAGE gel, transferred to the Immobilon membrane and probed with different purified nanobodies, followed by anti-His tag antibodies. (A) NbP1 detects a major 47–49 kD protein and a minor 110 kD protein. (B) NbP7, but not NbP8, detects a 210 kD protein. β-catenin levels indicate protein loading.</p

Estrogen-regulated protein EP45/Seryp is a target of NbP1.

<p>Semiconfluent HEK293T cells were transfected with the indicated DNAs. The cells were lysed 24 hrs later, and the protein lysates were separated on SDS-PAGE for imunoblot analysis with NbP1, followed by an anti-His tag antibody. NbP1 detects EP45 (arrowhead) in lysates of cells expressing pCS2-Ep45, but not pCS2-GFP. Asterisk points to a nonspecific band, which reflects loading. Embryo (stage 11) lysate is on the right, with two visible bands, as a positive control.</p

NbP1 immunoprecipitates a 49 kD protein from <i>Xenopus</i> embryos.

<p>A. NbP1 (P1) pull downs a specific protein from gastrula lysates (collected at st. 10.5–12). A normal embryo lysate (Lys) is shown on the left. NbP7 (P7) and no nanobody (-) groups represent negative controls. Immunoblot (probed with NbP1 and anti-His-tag) reveals heavy and light chains of the anti-His-tag antibody (asterisks), the nanobodies NbP1 and NbP7 (#) and the specific 47–49 kD protein band (arrowhead). B. Top candidate proteins detected by the LS-MS/MS analysis in a gel slice after immunoprecipitation by NbP1 from embryo lysates. Total scores based on the frequency of peptide representation, % coverage and molecular weights are indicated.</p

The carboxy-terminal fragment of Lulu functions as a dominant-negative mutant.

<p>(A) Top views of stage 9 embryos injected with indicated mRNAs. 100 pg of GFP-Lulu mRNA and 5 ng of GFP-Lulu-C or Flag-Lulu-C mRNAs were injected. Arrows point to ectopic hyper-pigmentation. (B) Percentage of the embryos showing ectopic hyper-pigmentation. Phenotypes were scored as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081854#pone-0081854-g004" target="_blank">Figure 4</a> legend. (C) Lysates from embryos injected with indicated mRNAs were subjected to Western Blot. α-tubulin serves as the loading control. (D) Sections of stage 9 embryos injected with indicated mRNAs were stained with anti-GFP and anti-flag antibodies. Superficial ectodermal cells are shown. Note the change of cell shape shown by the staining of GFP. Arrows indicate constricting cells. (E) Top views of stage 17 embryos uninjected or unilaterally injected with 5 ng of Lulu-C mRNA. Asterisks mark the injected side. Arrowheads point to the hinge of neural plate, and brackets indicate disappearance of the hinge. (F) Transverse sections of the neural plate of stage 17 embryos, unilaterally injected with GFP-Lulu-C mRNAs, were stained to visualize GFP, β-catenin, and F-actin. Dashed lines mark midline. Scale bar: 20 μm. </p

Depletion of Lulu disrupts apical constriction in neuroectoderm.

<p>(A, C-D) Transverse sections of the neural plate of stage 17 embryos unilaterally injected with 250 pg of GFP-CAAX mRNA and indicated MOs were stained to visualize GFP and (A) β-catenin, (C) F-actin by phalloidin, (D) phosphorylated myosin light chain (pMLC). Representative images are shown. In (C) and (D), exposure was adjusted to avoid oversaturation of apical staining. Arrows mark the injected side. Dashed lines mark midline. Scale bar: 20 µm. (B) Ratios of apical width to apico-basal cell length of superficial neuroepithelial cells were measured and compared. Up to five cells adjacent to midline were measured per section. Means +/- s. d. are shown. ***: p<0.0001, as compared to the LuluMO group. </p

The FERM and FA domains of Lulu are necessary for the induction of apical constriction.

<p>(A) Schematic illustration of various deletion mutants of Lulu. Amino acid numbers are indicated. (B) Embryos were injected with 250 pg of mRNAs encoding indicated Lulu mutants. Percentage of the embryos showing ectopic hyper-pigmentation at stage 9 was shown. Phenotypes were scored based on the degree of pigmentation increase, and representative images are shown on the right side. (C) Top views of stage 9 embryos injected with indicated mRNAs. (D) Sections of stage 9 embryos injected with indicated mRNAs were stained with anti-GFP and anti-β-catenin antibodies. Superficial ectodermal cells are shown. Note the change of cell shape shown by the staining of β-catenin. Scale bar: 20 μm.</p

MAK-V is not ubiquitinated by Nedd4.

<p>(<b>A</b>) E2 enzyme specificity of ubiquitin-ligase activity co-purified with MAK-V-FLAG protein. MAK-V-FLAG protein purified from PC12TetOn cells was used in <i>in vitro</i> ubiquitination reactions containing E1 enzyme and indicated E2 ubiquitin-conjugating enzymes. Results of Western blotting with HRP-conjugated streptavidin to detect ubiquitinated proteins/polyubiquitin (marked by asterisk) are shown. The image was overlaid with an image of the same membrane that was consequently probed with anti-FLAG antibodies to detect MAK-V-FLAG protein (marked by arrows). (<b>B</b>) Depletion of Nedd4 does not affect stabilization of MAK-V-FLAG protein in cells in response to treatment with proteasome inhibitors. Parental PC12TetOn cells with inducible MAK-V FLAG expression (<i>no miR</i>) or their clonal derivatives expressing control microRNA (<i>control mi</i>R) or microRNA to target Nedd4 transcript (<i>Nedd4 miR</i>) were treated with doxycycline to express MAK-V-FLAG protein and incubated with 100 µM of ALLN (<i>ALLN</i>) or 10 µM of MG132 (<i>MG132</i>) for 8 hrs or left untreated (-). Results of Western blot analysis of total cell lysates with anti-FLAG antibodies are shown (<i>anti-FLAG</i>). Nedd4 level was monitored with anti-Nedd4 antibodies (<i>anti-Nedd4</i>). MAK-V-FLAG protein marked with arrow, ubiquitinated higher molecular weight MAK-V-FLAG species marked by asterisk. Membrane was also probed with anti-α-tubulin antibodies (<i>anti-α-tubulin</i>) to monitor total protein loading. (<b>C</b>) MAK-V-FLAG protein and cytosols were prepared from parental PC12TetOn cells with inducible MAK-V-FLAG expression (<i>Nedd4</i>+) or its Nedd4-depleted clonal derivative expressing Nedd4 microRNA (<i>Nedd4</i> -) and used in <i>in vitro</i> ubiquitination reaction containing E1 and UbcH5b E2 enzymes. Results of Western blot analysis with anti-FLAG antibodies are shown. Arrow marks products of MAK-V ubiquitination. Reactions in the presence of EDTA (<i>EDTA</i> +) were used as negative controls.</p