The <i>LST1</i> exon 1B sequence inhibits protein expression.

<p>HeLa and HEK-293T cells were transiently cotransfected with expression constructs encoding the red fluorescent protein mCherry and either an EGFP expression vector, Exon1B-EGFP or Exon 1C-EGFP fusion construct. <b>(A, B)</b> Flow cytometry analysis of EGFP intensity in HeLa (A) and HEK-293T (B) transfectants expressing Exon1B-EGFP (yellow line), Exon 1C-EGFP (green line) or the unmodified EGFP vector (blue line). Untransfected cells are displayed as a solid grey curve. The mCherry expression was used to gate and select positive transfectants, which were analysed for the intensity of EGFP expression. <b>(C)</b> Lysates from HEK-293T transfectants were probed by western blot analysis using a GFP-specific antibody (lower panel). To ensure that comparable amounts of protein were loaded, the membrane was additionally probed with a tubulin-specific antibody (upper panel). <b>(D, E)</b> Quantitative flow cytometry analysis of EGFP expression in HeLa (D) and HEK-293T (E) transfectants. The analysis of EGFP expression was performed as described in (A, B) and the mean fluorescence intensity was quantified. A value of 100% was set for cells transfected with the unmodified EGFP vector. Mean values from 5 independent experiments are indicated within the columns +/− s.d. Both HeLa and HEK-293T transfectants expressing Exon1B-EGFP displayed significantly reduced EGFP levels when compared with cells transfected with the empty vector (p = 0.009). <b>(F)</b> Quantitative western analysis of EGFP expression. Lysates were probed as described in (C). The EGFP signal intensity was quantified and normalized for tubulin expression. A value of 100% was set for lysates from cells transfected with the unmodified EGFP vector. Mean values from 3 independent experiments are indicated within the columns +/− s.d. Transfectants expressing Exon1B-EGFP displayed significantly reduced EGFP expression levels when compared with cells transfected with the empty vector (p = 0.049).