Figure 7. Recombinant DsRed-tag54 and BP100-DsRed-tag54 accumulation in transgenic rice seedlings.

Western blot analysis of proteins from GM plants carrying: (A) p35S::hptII, pUbi::dsred-tag54, pHsp18::dsred-tag54 and pHsp82::dsred-tag54, either treated at 42°C (heat shock) or not (control); (B) pHsp82::bp100-dsred-tag54 (two independent events are shown) treated at 42°C. Recombinant protein in TSP was extracted from rice seedlings (five plants per event) and 20 µg of TSP per lane was boiled for five minutes and separated by SDS-PAGE before transfer to nitrocellulose filters. In lanes marked with an asterisk, insoluble pHsp82::bp100-dsred-tag54 pellets were re-extracted in 8 M urea and 1% SDS buffer. Recombinant proteins were detected using the mAb54k antibody (diluted 1∶1,500) and the horseradish peroxidase-labeled anti-mouse IgG secondary antibody (diluted 1∶10,000) followed by ECL chemiluminescent detection. Five to 20 pmol of chemically synthesized controltag54 mixed with 20 µg TSP from wild type rice was used as standards for quantification. GM rice carrying p35S::hptII was used as control. (C) Recombinant protein accumulation values. Means and SD of three independent events per construct are shown in filled boxes; letters indicate statistically different values; values corresponding to the highest producer event are shown in dashed boxes. (D) Western blot analysis of proteins from GM plants of a single event carrying pHsp82::dsred-tag54, either directly subjected to 42°C or with a 6 h initial step of gradual temperature increase up to 42°C (A+42°C) (five plants per condition). Recombinant proteins were extracted in 8 M urea and 1% SDS buffer. Senia (WT) was used as control.