Expression analysis of Mcoln1
Mouse adult multiple tissue and embryonic Northern blots were hybridized using a probe generated from mouse exon 2 (probe 1, Fig. 1B), yielding a band of approximately 2.4 kb (isoform 1), as expected, and a less abundant and unexpected 4.4 kb band (isoform 2) (Figs. 3A &3B). The 2.4 kb band shows ubiquitous but variable expression, with the highest expression in brain, liver and kidney. The fetal tissue blot shows decreasing levels of the 2.4 kb message with increasing gestational age. Since the human MCOLN1 gene encodes a single transcript [7-9], we carried out additional hybridizations with probes generated from various regions of the coding sequence and 3' UTR, and all probes identified the same two transcripts in the mouse (data not shown). In order to verify the presence of a single mouse locus, we hybridized a mouse Southern blot with the exon 2 probe. Four different restriction enzymes were used, and only the expected size bands for the chromosome 8 locus were detected (data not shown).
Figure 3  Northern analysis of Mcoln1. (A) Mouse fetal tissue Northern and (B) mouse multiple tissue Northern blots (Clontech) hybridized with a probe generated from exon 2 (probe 1, Fig. 1B). (C) Hybridization of the multiple tissue blot with a probe from the alternatively spliced segment of exon 13 (probe 2, Fig. 1B). β-Actin control hybridization is shown below each blot.