Figure 7. Confirmation of the identities of the endogenous ADAM12-La and ADAM12-Lb proteins detected in breast cancer cell lines.

(A) Sequences of two siRNAs designed to specifically target ADAM12 exon 4a (Ex4a) or exon 4b (Ex4b). The siRNA antisense strands are shown, and possible base pairing between each siRNA and Var-1a or Var-1b mRNA is indicated. The numbers refer to the nucleotide position in the coding sequence for each mRNA variant. (B) Efficacies and specificities of the two siRNAs. HEK293 cells stably expressing ADAM12-La or ADAM12-Lb were transfected with negative control siRNA (Ctrl), Ex4a siRNA, or Ex4b siRNA. Total cell lysates were analyzed by Western blotting using anti-ADAM12 antibody. β-actin is a loading control. (C,D) ADAM12-L knockdown in Hs578T and MCF10DCIS.com cells. Cells were stably transduced with lentiviruses bearing an shRNA construct targeting both ADAM12var-1a and ADAM12var-1b or control viruses. Alternatively, cells were transiently transfected with Ex4a siRNA, Ex4b siRNA, or negative control siRNA. Total cell lysates in C and D were enriched for glycoproteins on concanavalin A agarose prior to analysis. Full length ADAM12-La and ADAM12-Lb are indicated with solid and open arrowheads, respectively. The processed form of ADAM12-La is indicated with arrow. Epidermal Growth Factor Receptor (EGFR), ß-actin, and tubulin are gel-loading controls.