Visualisation was performed with amino ethyl carbasole (AEC) (Zymed Histostain kit) yielding a deep red colour.
The bound antibodies were visualized with the avidin-biotin peroxidase method (1 200, Vectastain Kit, Vector Laboratories), yielding a brown reaction product.
Eight replicates of each of the three dilution series was tested for each of the three kit lots, yielding a total of seventy-two repercates panelanel member.
The PWI mutants were constructed by conventional PCR with the p28-PWI plasmid as a template using the MutanBEST kit (TaKaRa), yielding a series of point mutations (K744A, H737A+K739A, K734A+R735A+K736A, R769A+R770A, K777A+K778A, K825A+R828A, K837A+K838A, K749A, R772A, K797A and K843A) in the p28-PWI plasmids and the plasmid with a deletion of the flanking basic region, ΔHb (Δ734 745).
The integrity of the RNA samples was determined with the BioAnalyzer (Agilent Technolgies, Amstelveen, The Netherlands) using the RNA nano 6000 kit (Agilent Technologies) yielding RIN-values ≥9.6.
PCR products were purified using Geneclean® Kit (Bio 101), yielding 15 μl solution of clean template in appropriate concentration for sequencing reactions with the ABI PRISM™ Dye Terminator Cycle Sequencing Kit PE Applied Biosystemss).
Successfully subcloned TFs (734) were miniprepped using the NucleoSpin 96 Plasmid miniprep kit (Macherey Nagel), typically yielding a concentration of approximately 150 300 ng/µl, from 2 ml overnight cultures in terrific broth II (MP Biomedicals, Santa Ana, CA).
meso-DAPDH gene was amplified using KOD -plus- DNA polymerase (Toyobo) with primers 5'-CACCATGAGTAAAATTAGAATTGGG-3' (forward) and 5'-TAAAAGTTCTTTTCTTAAATCTTCTGGAG-3' (reverse), and then cloned using a Champion™ pET 101 Directional TOPO Expression Kit (invitrogen, CA, USA), yielding the expression plasmid pET101/DAPDH.
CD4+ T cells were negatively selected with a CD4+ T-cell isolation kit (Miltenyi Biotec, Auburn, CA), yielding populations of CD4+ cells with the 96 99% purity.
