Transformation of Arabidopsis thaliana The pBCH1/dimeric hybrid-IgG/IgA was introduced into Agrobacterium tumefaciens GV3101 by electroporation selleck catalog using a Gene Pulser II (Bio-Rad, Hercules, CA, USA). Wild-type A. thaliana (ecotype Col-0) plants were grown in a temperature-controlled room with 24 h light at 20��C for 6 wk. A. thaliana plants were transformed via the floral dip method as described previously [34]. For efficient transformation, the floral dip procedure was performed 3 times at intervals of 3 or 4 d. The primary transformants were selected on MS medium supplemented with 20 ��g/ml hygromycin B. After selection, hygromycin B-resistant A. thaliana plants were transferred to soil in pots and then grown under the same conditions as for the wild-type plants.

The recombinant plants were grown within the physical containment level 1-plant (P1P) facility of the University of Shizuoka. DNA analysis Genomic DNA was extracted from the leaves of transgenic A. thaliana plants by boiling in a DNA extraction buffer (100 mM Tris-HCl [pH 9.5], 1 M KCl, 10 mM EDTA). PCR was performed with KOD FX Neo (TOYOBO, Osaka, Japan) using the following specific primer sets: IgG Heavy NotF and IgA-H/NotR for the heavy chain; IgGk NotF and IgGk NotR for the light chain; JCF-Xba and JCR for the J chain; and actin2-F and actin2-R for a house keeping gene ACTIN2 of A. thaliana. mRNA analysis Total RNAs were extracted from the leaves of transgenic plants using an RNeasy Mini Kit (QIAGEN, Hilden, Germany). RT-PCR was performed with an AccessQuick RT-PCR System (Promega) using the same gene-specific primer sets as for DNA analysis.

Protein extraction Leaves of transgenic A. thaliana plants were ground in liquid nitrogen to a fine powder with a mortar and pestle. Soluble proteins were extracted with a protein extraction buffer (10 mM MOPS-KOH [pH 7.5], 150 mM NaCl, 0.5 mM EDTA, protease inhibitor cocktail for plant cell and tissue extracts [Sigma-Aldrich, St. Louis, MO, USA]). Cell debris was removed by centrifugation (21,500 �� g, 10 min, 4��C), and the supernatant was used as a source of plantibodies. The protein concentrations in the extracts were determined with a BCA protein assay kit (Thermo Scientific, Rockford, IL, USA) using BSA (Thermo Scientific) as a standard. SDS-PAGE and immunoblotting Total soluble proteins were separated by SDS-PAGE on a 7.

5% gel (non-reducing conditions; Mini-PROTEAN? TGX? Precast Gels #456-1026, Bio-Rad) and a 12% gel (reducing conditions; Mini-PROTEAN? TGX? Precast Gels #456-1046, Bio-Rad), Dacomitinib and then electrically transferred to a PVDF membrane. Hybrid-IgG/IgA were detected with an SNAP i.d. Protein Detection System (Millipore, Billerica, MA, USA) with HRP-goat anti-mouse IgA (�� chain-specific) (1500 dilution; SouthernBiotech, Birmingham, AL, USA), goat anti-mouse kappa (2 ��g/ml; SouthernBiotech) plus HRP-donkey anti-goat IgG (0.