Skip to main content
Figure 1 | Cell Communication and Signaling

Figure 1

From: The importance of claudin-7 palmitoylation on membrane subdomain localization and metastasis-promoting activities

Figure 1

Claudin-7 palmitoylation: Impact on membrane subdomain localization and protein association (a-e) HEK cells were transfected with EpC and/or wt cld7 or EpC with a mutation of (mAG) in the transmembrane region or with cld7, with a palmitoylation site mutation. (a) WB of non-transfected and transfected HEK with anti-EpC, anti-cld7 and anti-actin (loading control); an example of flow cytometry analysis of EpC and cld7 in transfected HEK cells: single fluorescence overlays of negative controls and anti-EpC or anti-cld7 staining; (b) HEK-EpC-cld7 and HEK-EpCmAG-cld7 were lysed in the presence of N-ethylmaleimide to irreversibly block unmodified thiol groups. After incubation with HAM buffer for unmasking palmitoylated cysteine thiol groups, samples where incubated in biotin-BMCC for selective labeling palmitoylated cysteines. Samples were blotted with streptavidin-HRP and after stripping with anti-cld7. (c) WB of sucrose density fractions of untreated and 2-BP-treated HEK-EpC-cld7 and HEK-EpC-cld7mPalm with anti-cld7 and anti-EpC; (d) IP of HEK-EpC-cld7, HEK-EpCmAG-cld7 and HEK-EpC-cld7mPalm lysates with control IgG, anti-EpC and anti-cld7 and WB with anti-EpC and anti-cld7; (e) WB of non-mutated and mutated EpC and cld7 in light and heavy density fractions after density gradient centrifugation; constitutively GEM-located CD81 served as control; relative protein band intensity and the protein ratio in light to heavy fractions is indicated; (f) immunoprecipitation with control IgG, anti-EpC and anti-cld7 of lysates of non-mutated and mutated EpC and cld7 in light and heavy density fractions and WB with anti-EpC and anti-cld7; (g-i) SDS-PAGE, Coomassie-blue staining, in vitro kinase assay of whole cell and membrane lysates and WB with anti-p-tyrosine of anti-cld7 immunoprecipitates of HEK-EpC-cld7, HEK-EpCmAG-cld7 and HEK-EpC-cld7mPalm lysates. Co-transfection of HEK-EpC with cld7 shifts EpC into GEM. Instead, recovery of EpCmAG in GEM and co-immunoprecipitation with cld7 is strikingly reduced. Palmitoylation site mutated cld7 is hardly recovered in GEM and only few, mostly non-palmitoylated molecules associate with cld7mPalm.

Back to article page