Primary cell culture was conducted as previously described30,57. splicing variant (TFEB-S) lacking HLH and LZ regions of TFEB-L (Fig.?1A). To determine how the small TFEB was derived from the gene, we sequenced the small variant cloned into pHM6. Compared to the gene, the small CGP 36742 splicing variant was missing exon 8 (Fig.?1B). In addition, a nonsense mutation was introduced in exon 9 by a frameshift mutation due to an alternative splicing between exon 7 and exon 9 (Supplementary Fig. S1), generating a small TFEB protein of 281 amino acids (Supplementary Fig. S2). To confirm the isoform, we performed RT-PCR using a gene-specific primer set (Fig.?1C and Supplementary Table 1). As shown in Fig.?1D, a 214-bp PCR product was observed together with the main 290-bp fragment on a 1.2% agarose gel. To examine the expression of the small splicing variant in various tissues, we performed PCR using human cDNA panel prepared from various tissues. As shown in Supplementary Fig. S3, 214-bp PCR products were seen in most of human tissues. Together, the results suggest that the mRNA of small splicing variant is synthesized via an alternative splicing event in vivo. Open in a separate window Figure 1 A small TFEB splicing variant lacking the helix-loop-helix and leucine zipper (HLH-LZ) region is present in human cell lines. (A) A schematic comparison of TFEB-L and small TFEB (TFEB-S). glutamine rich, transactivation domain, nuclear localization signal, basic helix-loop-helix, leucine zipper, proline rich. (B) The small cDNA cloned into the pHM6 plasmid was sequenced by an automatic sequencer. Exon 8 was skipped in the small splicing variant. (C) The position of primers (Supplementary Table 1) used for RT-PCR of cDNA and the expected sizes of the respective PCR products in the presence and absence of exon 8 are shown. (D) RT-PCR was performed using cDNA prepared from HEK293 cells, and the PCR product CGP 36742 was analyzed on a 1.2% agarose gel. To examine the protein expression of the small splicing variant in various tissues, we performed immunoblotting of diverse proteins from human tissues. Approximately 30-kDa protein bands corresponding to small TFEB were observed in several tissues, with the highest expression level in the spleen and a moderate expression level in the brain. In contrast, the expression level of small TFEB was barely detectable in the liver and testis (Fig.?2). Open in a separate window Figure 2 A small TFEB protein is produced in various human tissues. CGP 36742 The expression level of TFEB and actin proteins was examined by immunoblotting using anti-TFEB and anti-actin antibodies, respectively. Full blots are provided in Supplementary Fig. S6. To examine whether small TFEB is expressed in different cell lines, we analyzed its expression levels in cellular extracts using immunoblotting. As shown in Fig.?3A, most of the cells, including primary-cultured rat cortical neurons (RCN), express small TFEB. Interestingly, the expression level of small TFEB was highly increased in T4 neuronal cells. In addition, we compared the mRNA levels of small TFEB to those of TFEB-L using qRT-PCR. The mRNA levels of small TFEB and TFEB-L varied by cell line. Specifically, the mRNA level of small TFEB was 18-fold lower in HEK293 cells than that of TFEB-L. In contrast, the mRNA expression level was 1,396-fold lower in T4 neuronal cells (Fig.?3B), suggesting that the splicing event producing the small variant might be uncommon in normal states. Open in a separate window Figure 3 A small TFEB protein is produced in various cell lines. (A) The expression level of TFEB and actin proteins was examined by immunoblotting using anti-TFEB and anti-actin antibodies, respectively. Full blots are provided in Supplementary Fig. S6. (B) The expression levels of TFEB-L and small TFEB (TFEB-S) mRNA Rabbit Polyclonal to FPRL2 in several cell lines were analyzed by qRT-PCR using specific primer pairs for each gene. Small TFEB is a.