NIR is associated with 32S and 12S pre-rRNAs and U8 snoRNA <i>in vivo</i> and knock down of NIR did not affect U8 snoRNA.

<p>A. Immunoprecipitation was performed with anti-NIR antibody on U2OS cell lysates, protein and RNA were prepared in parallel. Proteins were separated on SDS-PAGE, blotted onto a PVDF membrane and subjected to Western blotting analysis probed with anti-NIR (upper panel). Ten percent of cell lysates were loaded as input control. RNA extracted from the immunoprecipitation was resolved on 1% agarose-glyoxal gel and blotted onto a nylon membrane. Blot was probed with biotin-labeled ITS-2 oligonucleotide (lower panel). B. Immunoprecipitation was performed with anti-NIR antibody and proteins from the immunoprecipitation were subjected to Western blotting analysis probed with anti-NIR as described in A (upper panel). Ten percent of cell lysates were loaded as input control. RNA extracted from the immunoprecipitation was resolved on 7% polyacrylamide/8.3 M urea and blotted onto a nylon membrane. Blot was probed with biotin-labeled U3 snoRNA-specific RNA probe or U1-specific RNA probe (lower panel). RNA extracted from 2.5% cell lysates were loaded as input control. C. U2OS cells were transfected with NIR specific siRNA (siNIR) or a siRNA targeting luciferase (siLC) as a control. Seventy-two hours post transfection, total RNA was extracted. Equal amount RNA was resolved on a 7% polyacrylamide–8.3 M urea gel and transferred onto a nylon membrane. Blot was hybridized with U8 snoRNA specific probe.