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We use BLAST search (http://www.ncbi.nlm.nih.gov/BLAST) to check the specificities and homology percent of primers for the VDR sequence.
To check the specificities of A6RPT- 5803 and 2D-1, immunoblot procedures were undertaken in a special device (ATTO, Tokyo, Japan) that makes it possible to apply different antibodies to one blotted membrane at a time.
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To check the specificity of linkage-specific anti-ubiquitin antibodies, Lys 48- or Lys 63-linked polyubiquitin chain (Enzo Life Sciences) was used as a positive control and subjected to immunoblotting.
After PCR amplification, a melting curve was generated to check the specificity of each PCR reaction (absence of primer dimers and other non-specific amplification products).
Moreover, MRM spectra of blanks with mass fragmentation patterns of CVA and IS were obtained to check the specificity of the method.
Eighty four other bacterial strains, either commonly occurring in sheep or found in the environment of sheep, and including organisms related taxonomically to D. nodosus, were used to check the specificity of these assays.
Blocking peptides were also used to check the specificity of every antibody (data not shown).
After amplification, a melting curve was acquired to check the specificity of PCR products.
Mutated sequences of the transcription factors were also used to check the specificity of the binding of the transcription factors.
The anti-p65 (p65α) antibody was added to check the specificity and cold probe served as a negative control.
To check the specificity of the designed primers, all amplicons were sequenced and blasted against NCBI database.
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