JAG2 expression. (A) FACS analysis of normal (left panel) and MM (right panel) plasma cells for JAG2 expression. Shown are the JAG2 expression profiles of the CD138⁺ plasma cells using either a goat Ig isotype or anti-JAG2 primary antibodies. (B) Western blot analysis of 4 MM (RPMI 8226, U266, MM1-S and -R) and 1 lymphoblastoid (EBV-LIN) cell lines for the Notch-1, -2, -3, -4, and Jagged-1 proteins with GAPDH as loading control. (C) Time-course Western blot on the RPMI 8224 cell line (MM, JAG2-positive) (top panel) and the NIH 3T3 cell line constitutively expressing a retrovirally transduced JAG2 (bottom panel) after incubation with puromycin. No difference in JAG2 protein stability is detected between the two cell lines. If a stabilized protein were to be present in the MM cell lines, the JAG2 protein would be still present after the production of new protein has been blocked by puromycin. (D-F) Physical mapping of the JAG2 gene. (D) National Center for Biotechnology Information (NCBI)–related mapping showing the proximity of JAG2 with IgH and (E) confirmation of the physical mapping using the RP11-336L21 BAC clone in combination with the Ig10 cosmid on normal chromosome metaphase. (F) Fiber-FISH profile obtained with extended DNA from both normal controls and MM cell lines using the RP11-336L21 and the Ig10 probes.