FIGURE 2.

Characterization of anti-DCT and anti-CAV1 antibodies in Western blot and immunofluorescence microscopy. MJS (72 h) and SK28 (96 h) melanoma cell lines transfected with siRNA control (si-ct), si-DCT (si-DCT), or si-CAV1(si-CAV1) were assessed for DCT and CAV1 expression in Western blot (WB) and immunofluorescence microscopy (IF) with D18 or α-hDCT and α-CAV1-CS or α-CAV1-sc, respectively. WBs were assessed with ECL or SuperSignal West Femto chemiluminescent substrate (Femto) detection systems. Both anti-DCT and anti-CAV1 antibodies recognize specifically in the two cell lines the products encoded by the DCT and CAV1 gene, respectively, different CAV1 constituents (monomers, oligomers, in Nonidet P-40-soluble (sol) and Nonidet P-40-insoluble (insol) fractions). Importantly, in both cell lines, an insoluble oligomeric CAV1 pool detected with both anti-CAV1 antibodies was resistant to CAV1 down-regulation. Unlike in MJS, the soluble oligomeric CAV1 detected preponderantly with α-CAV1-sc in SK28 cells was also resistant to si-CAV1 indicating that CAV1 aggregation is different in the two cell lines. The nonspecific DCT and CAV1 bands are indicated by stars. Calnexin (Clx) was used as loading control for Nonidet P-40-soluble fractions. The IF images were acquired using ×40 objective. Scale bar represents 10 μm. Each experiment is a representative one of three.