The cryptic promoter mediates expression of the short spastin isoform in vivo. (a) HeLa cells were transfected with a CMV-spastin-GFP, a CMV-spastin-ΔM1 or a spastin-GFP- ΔCMV construct. Cell lysates were prepared 48 hours post-transfection and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Immunoblotting for transfected spastin was performed with the S51 polyclonal antibody. The CMV-spastin-GFP plasmid drives expression of two spastin isoforms starting from the first and second methionine, as previously described . Consistently, the CMV-spastin- ΔM1 construct produces only the shorter isoform. Notably, the promoter-less spastin construct synthesizes the short isoform, albeit at lower level, indicating that the cryptic promoter of spastin is active in vivo. Below each lane, the amount of transfected cell lysate loaded is indicated. (b) Similar results were obtained in the murine immortalized motoneuronal cell line NSC34. (c) The empty CMV-EGFP vector and CMV-EGFP-STOP-spastin construct were transfected in HeLa cells. Immunofluorescence was performed 48 hours after transfection. Transfected cells were detected by enhanced green fluorescent protein epifluorescence, while spastin was revealed using the S51 polyclonal antibody. Cells expressing high levels of GFP also synthesize low levels of spastin. Note the different pattern of GFP (diffuse) and spastin staining (discrete, as described previously ).