Distribution of <i>LvPT</i> in different tissues by semi-quantitative PCR.

<p>Ten tissues from 5 individuals were collected and the distribution of <i>LvPT</i> was analyzed by semi-quantitative PCR. The expected length of <i>LvPT</i> and 18S rRNA fragments were 222 and 166 bps, respectively.</p

Interactions between LvPT and VP32, VP38A, VP39B, and VP41A.

<p>The blue clones on QDO/X/A plates (SD/-Ade/-His/-Leu/-Trp supplemented with X-a-Gal and Aureobasidin A) represent interactions between LvPT and VP32 (A), VP38A (B), VP39B (C), and VP41A (D). The numbers around the plate indicate the bait and prey plasmids of the transformed yeast: 1, pGBKT7-53/pGADT7-T (positive control); 2, pGBKT7-VPXX/pGADT7—LvPT1; 3, pGBKT7/pGADT7-LvPT1; 4, pGBKT7-Lam/pGADT7-T (negative control).</p

Confirmation of interaction between LvPT and VP37 on QDO/X/A plate.

<p>The blue clones on QDO/X/A plates (SD/-Ade/-His/-Leu/-Trp supplemented with X-a-Gal and Aureobasidin A) represent interactions between LvPT1/ LvPT2 and VP37. The numbers around the plate indicate the bait and prey plasmids of the transformed yeast: 1, pGBKT7-53/pGADT7-T; 2, pGBKT7-37/pGADT7-LvPT1; 3, pGBKT7-37/pGADT7-LvPT2; 4, pGBKT7-Lam/pGADT7-T; 5, pGBKT7/pGADT7-LvPT1; 6, pGBKT7/pGADT7-LvPT2</p

Analysis of the quality of inserts in the Y2H library.

<p>Lane M indicates the D2000 II DNA ladder (Real-Times Biotechnology), while lanes 1–20 indicate the size of insert fragments of 20 clones randomly picked from the SD/-Leu plate.</p

Predicted domains of LvPT1 (A) and LvPT2 (B).

<p>Amino acid sequence analysis of LvPT1 and LvPT2 revealed the presence of three conserved ChtBD2 domains; in addition, a signal peptide was observed at residues 1 to 19.</p

Map of pGADT7-DEST.

<p>This plasmid was modified from pGADT7-Rec (Clontech Laboratories); the SMART III and CDS III sequences were replaced by attR1 and attR2, respectively. Following recombination catalysis using the Gateway^® LR Clonase^™ II enzyme mix (Invitrogen), attR1-Cm-ccdB-attR2 was replaced by attB1-cDNA insert-attB2.</p

Multiple sequence alignment of LvPT1, LvPT2, and peritrophin [<i>Litopenaeus vannamei</i>].

<p>The underlined sections indicate conserved ChtBD2 domains in LvPT1, LvPT2, and peritrophin, and the shaded sections in each ChtBD2 domain indicate the presence of cysteine residues.</p

LvPT-V5 and LvPT^WSP-V5 expression in Sf9 cells.

<p>Sf9 cells were transfected with plasmids expressing LvPT-V5 (V5-tagged LvPT) and LvPT^WSP -V5 (LvPT-V5 without signal peptide). At 6 h after heat shock, the culture medium, supernatant and precipitate of cell lysates were harvested. After separation by SDS-PAGE, LvPT-V5 and LvPT^WSP -V5 were detected by western blot using anti-His antibody as a probe. Asterisks indicate the LvPT-V5 while triangles indicate LvPT^WSP -V5 in samples. Compared with LvPT^WSP-V5, LvPT-V5 could be detected in culture medium and tended to be a secretory protein in Sf9 cells.</p

Results of co-immunoprecipitation.

<p>Sf9 cells were transfected with plasmids expressing VP37-FLAG (FLAG-tagged VP37), LvPT-V5 (V5-tagged LvPT) or empty plasmid (vector). At 6 h after heat shock, the cell lysates were harvested. (A) After separation by SDS-PAGE, expression of VP37-FLAG and LvPT-V5 was confirmed by western blot using anti-His antibody as a probe. Arrows indicate the expressed VP37-FLAG and LvPT-V5. (B) The cell lysates were immunoprecipitated with anti-FLAG M2 affinity resins and then the immunoprecipitated complexes were subjected to western blot analysis with anti-His antibody probe.</p

Chitin binding assay of LvPT.

<p>LvPT-V5 was incubated with colloidal chitin at 4°C for 2 h, in order to eliminate dissociative proteins, the LvPT-V5-chitin complex washed three times by TBS (50 Mm Tris, pH 8.0) supplemented with 50 mM NaCl, 100 mM NaCl, 200 mM NaCl and 500 mM NaCl, respectively. The LvPT-V5 bind to chitin (bands around 40 kDa) was detected by western blot using anti-His antibody as a probe. BSA was used as negative control and detected by coomassie brilliant blue. Colloidal chitin (protein-free) was used as reference to indicate the bands in negative control were not proteins but polymer of N-acetyl glucosamine.</p