Al PDZ binding domain (Itoh et al., 1999; Michlig et al., 2007). No interaction was observed between claudin-8 and ZO-1 (PDZ2-3) as expected; even so, G13 connected weakly with ZO-1 (PDZ2-3)PS10 custom synthesis Frontiers in Cellular Neurosciencewww.frontiersin.orgJune 2012 | Volume 6 | Post 26 |Liu et al.ZO-1 interacts with GFIGURE two | Expression of GOPC, MPDZ, ZO-1 and G13 in circumvallate papillae. (A) RT-PCR experiment demonstrating expression of ZO-1, GOPC, and MPDZ in all tissues tested. In contrast, the presence of G13 and Ggust (GUST) mRNAs seem to become restricted to taste and olfactory sensory tissues. See Section “RT-PCR” and Table A1 for particulars about amplification conditions and expected sizes of PCR goods. GAPDH primers were used as a control from the good quality of the RNA. (+) and (-) indicate the presence or absence of reverse transcriptase in the reaction respectively. L: 100 bp Demecycline custom synthesis ladder. NT, tongue epithelium deprived of taste buds; CV, circumvallate papillae; OE, olfactory epithelium; Br, whole brain; Liv, liver. The results presented are representative of three independent experiments. (B) Immunodetection of ZO-1, MPDZ, GOPC, and G13 proteins in circumvallate (CV) and entire olfactory epithelium (OE) protein extracts. Sample preparation and immunodetection conditions following western blotting are as described in detail beneath Section “Co-immunoprecipitation and Western blotting.” Protein extracts from HEK 293 cells (HEK) or HEK 293 cells stably expressing HA-G13 transiently transfected using a full length Myc-ZO-1 construct (HEK ZO-1 13) were utilised as controls. As expected ZO-1 and G13 are detected in CV and OE. Note that G13 displays a higher apparent molecularweight in CV than in OE. Predicted molecular masses for ZO-1, MPDZ, GOPC, G13, and -actin are 220, 220, 51, 8, and 42 kDa respectively. -actin was utilised as loading control. The results presented are representative of three independent experiments. (C) Localization of MPDZ, GOPC, G13, and ZO-1 proteins in circumvallate taste buds sections. Indirect immunofluorescence on longitudinal cryosections of circumvallate papillae was performed as described under Section “Immunohistochemistry.” MPDZ, GOPC, and G13 are primarily distributed inside the cytoplasm of a subset of taste bud cells though ZO-1 localizes mainly in the taste pore. Around the Nomarski image a white dashed line highlights the size and place of 1 taste bud. G13, MPDZ, and GOPC pictures are strict confocal optical sections (pinhole 82 m, airy disk 1) whilst a wider pinhole was utilized for the ZO-1 image (pinhole 124 m, airy disk 1.7), Scale bar = 50 m. Staining patterns are representative of two independent experiments performed on multiple sections from at the very least two mice. (D) Drawing representing a longitudinal section in the circumvallate papillae as in (C), displaying the place with the taste buds along the walls of your trench beneath the surface of the tongue. Taste bud cells protrude into the trench via the taste pore.Frontiers in Cellular Neurosciencewww.frontiersin.orgJune 2012 | Volume six | Short article 26 |Liu et al.ZO-1 interacts with GFIGURE 3 | Physical interaction among heterologously expressed G13 and ZO-1. (A) To test the strength of the interaction amongst the proteins assayed Mav203 yeasts cells have been co-transformed with diverse combinations of bait and prey plasmids (see crucial). Interaction was scored on minimal medium plates lacking His, Leu, and Trp but containing increasing concentrations of 3-AT (00 mM) a competitive inhibi.
