Single point mutations were introduced into wild-type or mutant cDNAs using the QuikChange® site-directed mutagenesis protocol (Stratagene) and the coding regions of each mutant were fully sequenced.
A few of the SOD1 mutants cDNAs used have been previously created into the pEF-BOS system (WT, A4V, G37R, G41D, G85R, G93A and G93C SOD1) (23, 51); however, many other mutants were kindly provided, in the YEp351 yeast vector, by Dr Stephen Holloway (University of Texas Health Sciences Center, San Antonio, TX, USA), which we subcloned into our pEF-BOS mammalian vector.
Comparing hybridisation intensity between control and mutant derived cDNAs, using cut off points of at least a 2-fold change and corrected p-value ≤0.05 [ 27] for significance across replicates, identified 374 independent genes as differentially expressed (DE).
For this, total RNA from ISE6 tick cells infected with A. marginale wild-type and omp10::himutantutant was reverse transcribed and cDNA used as template for PCR amplification with specific primers that were designed to anneal to omp6, omp7, omp8, omp9, and omp10 in wild-type and omp10::himutantutant respectively.
A limitation of the transfection assay is that mutant cDNA constructs were used and therefore the experiments did not evaluate the impact of NMD upon mutant protein expression.
Viral RNA transcripts from E7 WT and mutant cDNA clones were used to program rabbit reticulocyte lysates in the presence of [S]-methionine.
We derived the Flag-Cdc42N17 and myc-Rac1N17 mutant cDNAs by PCR amplification using PFU polymerase (Stratagene, La Jolla, CA), and separately subcloned each into the pTetPuroR vector.
The next day, cells were transiently transfected with mutant forms of mGlu5 cDNA using FuGENE 6 (Roche Applied Science, Indianapolis, IN) according to the manufacturer's instructions (2 μg of DNA, 24 μL of FuGENE 6, and 776 μL of Opti-MEM (Invitrogen, Carlsbad, CA)).
Peripherin 45 mutant was generated from peripherin cDNA using the following oligonucleotides: 5′-ATAGCGATCGCCATGGCCGAGGCCCTCAAC-3′ and 5′-CCGCGTACGCGTGTAACTGTGGGC-3′ and then cloned with AsiSi and MluI restriction enzymes into the myc-tagged vector.
The pPGK-IRES-p40 plasmid was used to express some RFM mutant cDNAs from a bicistronic message; this vector has been described previously [16].
