Figure 10.
Suppression of Sec3 expression is associated with increases in overall expression levels of desmosomal cadherins and plakoglobin. (A) Two clonal populations of MCF-10A cells stably transduced with shSec3 (hairpin 2) were isolated and characterized. Sec3 and β-tubulin immunoblots were performed to determine the level of Sec3 reduction (values for each clone are normalized to control cultures). Note that clone 1 exhibited an almost 80% reduction of Sec3 and that clone 2 exhibited a >90% reduction. (B) Lysates of control MCF-10A or shSec3 clones 1 and 2 were immunoblotted with antibodies to desmosomal cadherins (Dsg1, Dsg2, and Dsc2/3), plakoglobin (PG), DP1/2, and β-tubulin. Dsg1 and 2, Dsc2/3, and plakoglobin protein levels were all increased in the Sec3 knockdown clones, relative to controls. DP1/2 protein levels did not change. (C and D) Immunoblots of two adherens junction-associated proteins, E-cadherin and α-catenin, showed no consistent changes in expression levels in the Sec3 knockdown clones. (E) Lysates of MCF-10A cells, shSec3 cells, or shSec3 cells transiently transfected with a plasmid encoding a hairpin-resistant variant (Sec3hr) were immunoblotted for Dsc2/3, PG and β-tubulin. Note that reexpression of Sec3 in cells partially reversed the elevation of desmosomal protein expression that accompanied Sec3 knockdown, as normalized levels of both Dsc2/3 and PG were lower in cells that received the Sec3 rescue construct.