Fig. 2. (A) Incorporation of [³H]-palmitate into endogenous claudin-2 in MDCK cells. MDCK cells were labeled with [³H]-palmitic acid, extracted and immunoprecipitated. Samples were divided in half and electrophoresed on SDS-PAGE gels in parallel; one gel was transferred to nitrocellulose and immunoblotted for claudin-2 and the second was treated with Amplify fluorographic reagent (Amersham), dried and exposed to Hyperfilm MP for 3 weeks. (top) Immunoblot for claudin-2. NS refers to a nonspecific immunoprecipitate. (bottom) Signal from ³H-palmitate incorporation into claudin-2. Incorporation of ³H-palmitate into claudin-14 and CS mutants. MDCK cells were stably transfected with wild-type and mutant claudin-14 with the VSV-G tag and transgene expression induced by removal of doxycycline. After 48 hours of induction, cells were labeled, extracted and immunoprecipitated as described above, but with a VSV-G monoclonal antibody. Lanes: (+), with doxycycline (not induced); WT, wild-type claudin-14; CTM2S, CysSer mutation after the second transmembrane domain; CTM4S, CysSer mutation after the fourth transmembrane domain; 4S, CysSer mutation after the second and fourth transmembrane domains. (top) Immunoblot for VSV-G. (bottom) Signal from the incorporation of ³H-palmitic acid into the immunoprecipitated protein; this experiment was repeated with similar results. The faint band in the immunoblot above the claudin/VSV-G band is nonspecific signal from immunoglobulin light chain.