This hypothesis was further supported by the finding that ZNF9 can bind ribosomal protein mRNA in Xenopus and, more recently, in humans [42,43]. Moreover, recent studies show that ZNF9 is part of a ribonucleoprotein complex that promotes cap-independent mRNAs translation [44]. Western blot analysis presented here indicates that: (i) the K20 Ab, used in the subsequent experiments on ZNF9 localization, recognizes a single electrophoretic band consistent with ZNF9 MW (19 kDa) in rat and human tissue extracts; and (ii) ZNF9 is ubiquitously expressed in mammalian tissues, at the highest level in liver, spleen

and brain, and at a lower level in heart and skeletal muscle. This last result is not entirely consistent with the tissue distribution of ZNF9 mRNA observed Seliciclib in a recent report [24]. The discrepancy could be due to tissue-specific translational and/or post-translational H 89 manufacturer regulation, which would be interesting to further investigate.

In addition, our WB analysis revealed that the signal of ZNF9 does not appear to be consistently altered in DM2 muscles as compared with normal, although some variability was observed. We obtained similar results probing DM2 lymphoblastoid cells with the antiserum from which the K20 Ab was purified [38]. Normal levels of ZNF9 mRNA and protein were also detected by Margolis et al.[45] in myoblasts and muscle tissue from heterozygous and homozygous DM2 patients using an Ab to the middle portion of the ZNF9 protein. On the other hand, two recent studies report a decrease of ZNF9 protein in DM2 myoblasts and muscle

biopsies [42,46]. Several reasons that may underlie this discrepancy may include the presence of mixed cell populations in biopsies as opposed to the purity of myoblast culture, the use of different cell types (lymphoblastoid vs. myoblasts) or different Abs. Moreover, the limited number of samples used in this and in other studies suggests that more definitive data on ZNF9 expression in DM2, possibly correlated with histological grading and [CCUG]n expansion size, should be obtained from larger pools mafosfamide of patients. Our IF experiments are helpful in locating ZNF9 in myofibres, in relation to subcellular structures. The combination of a myofibrillar pattern of distribution in transverse section, and the localization to cross-striational bands with a thickness of about 1 µm, corresponding to the size of I bands in semi-relaxation, suggests a location of ZNF9 immunoreactivity within or in association with sarcomeric structures. This is confirmed by the results obtained from double IF experiments. Indeed, when comparing ZNF9 distribution with that of two non-repetitive epitopes located at distant sites along the titin molecule, we observed different patterns of localization.