The infected calli were successively cultured in DKN selection, DKN regeneration, and MS regeneration media with hygromycin (Wakasa for 20 min at room temperature. a higher-order molecular form different from the normal mature glutelins. Materials and methods Herb materials Rice (L.) cv. Koshihikari mutant line a123 (Iida are lacking because of mutation, was used for transformation. Vector construction Gene cassettes consisted of the endosperm-specific 2.3 kb glutelin B1 (terminator (Accession No. “type”:”entrez-nucleotide”,”attrs”:”text”:”X54314″,”term_id”:”20209″,”term_text”:”X54314″X54314) were introduced into the multiple cloning sites of modified-pBluescript KS+ made up of the Gateway recombination sites att L1 and att L2. GSK-3 inhibitor 1 Then, two gene cassettes were transferred from entry clones to a destination-binary vector (p35:HPT Ag7-GW; Wakasa gene 7 terminator; HPT, hygromycin phosphotransferase coding region; 35S pro, CaMV 35S promoter; 2.3 K GluB1 pro, 2.3 kb rice glutelin B1 promoter; GluA2, wild-type rice glutelin A2 coding region; GluB ter, 0.65 kb glutelin B1 terminator; mGluA2, mutated-glutelin A2 coding region. Production of transgenic plants Transgenic rice plants were produced by strain EHA105 by electrotransformation. Four-week-old calli derived from mature seeds were co-cultured with Adam23 the transformed for 3 d. The infected calli were successively cultured in DKN selection, DKN regeneration, and MS regeneration media with hygromycin (Wakasa for 20 min GSK-3 inhibitor 1 at room temperature. Proteins were subjected to immuno-blot analysis using anti-GluA (GluA1 and GluA2), anti-GluB (GluB1, GluB2, and GluB4), anti-BiP, anti-PDI, and anti-calnexin antibodies. For quantitative dot blot immuno-blotting analysis, four positive seed extracts per impartial transgenic line selected by immuno-blot using anti-GluA antibody were pooled and used for quantification. For the relative comparison of transgene product accumulation, equal amounts of the protein extracts were spotted onto a nitrocellulose membrane (Whatman, Dassel, Germany). The transgene products in each dot were detected immunologically with anti-GluA antibody and quantified with NIH Image J (National Institutes of Health ver. 1.41; Washington, DC, USA). Sequential protein extraction GSK-3 inhibitor 1 Sequential extraction of proteins was performed according to Takaiwa (2008). Briefly, globulins were extracted with 500 l of globulin extraction buffer (0.5 M NaCl, 10 mM TRIS-HCl, pH 6.8) from 25 mg seed powder. After the removal of globulins, the glutelins were extracted from residual proteins with 500 l of glutelin extraction buffer (1% lactic acid, 1 mM EDTA). Each extraction step was repeated five occasions and accomplished by sonication for 2 min and centrifugation at 13?000 for 10 min. After the stepwise removal of the globulin and glutelin fractions, prolamins were finally extracted from residual proteins with 500 l of total protein GSK-3 inhibitor 1 extraction buffer. To examine the possible involvement of disulphide bonds for extraction efficiency, the globulin fraction was extracted from 25 mg seed powder with 500 l of globulin extraction buffer with or without 5% 2-MER. After the acetone precipitation, the globulin pellet was dissolved with 100 l of total protein extraction buffer. The residual proteins were extracted with 500 l of total protein extraction buffer. Two microlitres of each sample were used for SDSCPAGE and immuno-blotting analysis. Immunogold electron microscopy The immature seeds (15C20 DAF) were fixed in 4% paraformaldehyde, 0.1% gultaraldehyde buffered at pH 7.2, with 20 mM PIPES over night at 4 C. After washing in PIPES buffer, the samples were dehydrated in a series of ethanol concentrations GSK-3 inhibitor 1 and embedded in LR White resin (London Resin, Berkshire, UK). Ultrathin sections were cut with a glass knife using an ultramicrotome (MT2-B; Sorvall, Newtown, CT, USA) and mounted on copper grids. The grids were floated on a drop of PBS made up of 3% BSA for 1 h and were incubated for 1 h with anti-GluA diluted 1:3000 with PBS made up of 1% BSA. Nonspecifically bound antibodies were removed by washing the grids with washing buffer (PBS made up of 0.1% BSA,.