Figure 1. Characterization of ASML-EpC and cld7 knockdown and rescue clones.

ASML cells were transfected with EpC- and cld7-shRNA and cloned in selection medium. EpC expression was rescued in ASML-EpC^kd clones using primers for wt rescue (EpC^resc) or point mutated (position 282 and 279) EpC (EpC^mAG); cld7 was rescued in ASML-cld7^kd clones with a mutation at the palmitoylation site at AA184 and AA186 or was transiently rescued using primers for wt rescue (cld7^resc). (A) Wt, kd and rescue ASML clones were lysed in the presence of N-ethylmaleimide (NEM) 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 of palmitoylated cysteines. Samples were blotted with streptavidin-HRP and after stripping with anti-cld7; (B) lysates of wt, kd and rescue ASML clones (one representative clone was selected) were separated by SDS-PAGE and blotted with anti-EpC (D5.7), anti-cld7 and anti-Tspan8 (D6.1, control); (C) lysates of wt, kd and rescue ASML clones were precipitated with anti-EpC or anti-cld7. After SDS-PAGE, precipitates were blotted with anti-EpC, anti-cld7 or anti-Tspan8; (D) wt and rescue ASML clones were stained with anti-EpC(red) and anti-cld7 (green); staining was evaluated by confocal microscopy, digital overlays (scale bar: 10 μM). The indicated area (white square) was amplified 10-fold for better discrimination. The Pearson correlation coefficiency is shown for the encircled membrane area; (E) lysates of wt, kd and rescue ASML clones were separated according to density by sucrose gradient centrifugation; 1ml fractions were collected and fractions 5–8 and 9–12 were pooled. After SDS-PAGE, fractions were blotted with anti-EpC, anti-cld7 and anti-Tspan8 (GEM control). EpC and cld7 were efficiently downregulated in kd clones and were recovered in rescue clones. Mutation of the palmitoylation site at AA184 and AA186 prevented cld7 palmitoylation. Cld7 palmitoylation strongly facilitates the association with EpC, the cld7-EpC complex being enriched in GEM.