Inhibition of the Sec insertion machinery, or ribosomal pausing may not be observable in the luciferase system. As there is no simple way to individually detect both the Seccontaining full-length SelS protein and a two amino acid truncated form, a V5 epitope tag was introduced between the UGA codon and the UAA stop codon. The V5 tag is easily detectable and in these Lomitapide Mesylate constructs the expression of the V5 tag is dependent on Sec insertion, as termination at the UGA codon would Tulathromycin B prevent inclusion of the tag. The above results demonstrate that the potential exists to produce two different SelS protein isoforms, a full-length protein containing a penultimate Sec residue and a truncated protein that does not contain 
Sec. We wondered whether the different carboxy-terminal ends would affect the subcellular localization of the protein. There are several examples where exposed thiols have been shown to be important for ER localization of proteins by mediating intramolecular bonds. In addition, a precedent exists for a penultimate cysteine being required for the ER retention of the secreted immunoglobulin M heavy chain. SelS is a membrane protein and was previously shown to localize to the ER and plasma membrane by overexpression of epitope-tagged SelS constructs or fractionation experiments. Given the availability of a suitable antibody for immunofluorescence, we examined endogenous SelS localization. SelS is predominantly found in the ER, with some weak staining of the plasma membrane in some cells. More strikingly, there is an accumulation of SelS in a perinuclear region. This localization is not cell type specific as we observed similar results in U251MG and HepG2 cells. It is also not an artifact generated during the fixation step as acetone, methanol and 4% paraformaldehyde methods all showed this accumulation. Previous studies would not have observed this localization as the overexpressed SelS obscures this perinuclear signal. Given that the Golgi apparatus often shows a similar staining pattern, we concurrently stained the cells for endogenous SelS and a Golgi marker. As shown in Figure 8B, colocalization of these two proteins was detected next to the nucleus. In order to examine this potential colocalization more carefully, the cells were examined by confocal microscopy. A series of focal planes that spanned the depth of the cell were examined for SelS and golgin p97 localization. As shown in the image gallery, there is some spatial overlap between the two proteins, but it is not a complete colocalization. In order to address whether these ER and perinuclear localizations might represent the two different SelS proteins, we treated HepG2 cells with siRNAs directed against both SelS isoforms, as well as variant 1 and variant 2specific siRNAs. Localization of endogenous SelS protein was examined by immunofluorescence after siRNA treatment. When treated with siRNAs that target both SelS mRNA variants, the punctate perinuclear signal persists, after the ER localization is no longer detectable. A similar staining pattern was observed using siRNA directed solely against the variant 2 transcript. In contrast, cells treated with the siRNA against transcript variant 1 looked similar to cells treated with a nontargeting control siRNA. Similar results were obtained with U251 cells. Thus, the ER and perinuclear localizations are not simply due to two different protein isoforms from the variant mRNA transcripts.