Figure 3. CERKL kinase activity assays. A: The autoradiography of one out of many thin layer chromatographies (TLC) from in vitro assays is shown. Lipid micelles and heat-inactivated cell lysates from 661W (murine photoreceptor-derived cell line) cells were used as substrates, whereas protein lysates from COS-7 cells transfected with either empty vector (pcDNA), CERKL isoform a (532 aa), CERKL isoform b (558 aa) or ceramide kinase CERK (positive control) expressing constructs were the source of the enzymatic activity. B: Autoradiography of a TLC from in vivo assays is shown. Cultured 661W cells were transfected with either pGFP (empty vector), or CERKLa-GFP, CERKLb-GFP, or CERK-GFP (positive control), selected by FACS and grown in a medium supplemented with ³²P-orthophosphate (see Material and Methods for details on the protocols). C1P represents Ceramide-1-phosphate. The images are representative of many different assays, with several experimental parameters, such as the type of cell line and the CERKL isoforms overexpressed, changed. The results of the assays were negative under all the conditions tested.