Aberrant expression and subcellular localization of MCC in human MM cells. (A) Taqman assay of the MCC transcript. Total cellular RNA was prepared from normal human blood B lymphocytes (Normal B), an EBV-transformed B lymphoblastoid cell line C3688, or human MM cell lines with TRAF3 deletions or relevant mutations. The human MM cell lines include KMS11, 8226, LP1, U266, KMS28PE, and KMS20. Real-time PCR was performed using TaqMan primers and probes (FAM-labeled) specific for human MCC. Each reaction also included the probe (VIC-labeled) and primers for human β-actin mRNA, which served as an endogenous control. Relative mRNA expression levels of the MCC gene were analyzed using the Sequencing Detection Software (Applied Biosystems) and the comparative Ct (ΔΔCt) method. Graphs depict the results of three independent experiments with duplicate reactions in each experiment (mean ± S.D.). (B) Western blot analysis of the MCC protein. Total cellular proteins were prepared from normal B lymphocytes, C3688 cells, or human MM cell lines with TRAF3 deletions or relevant mutations. Proteins were immunoblotted for MCC, followed by TRAF3 and actin. Data shown are representative of 3 experiments. (C) Subcellular localization of MCC and its regulation during ER stress responses. LP1 cells were cultured in the absence (control) or presence of ER stress inducers DTT or thapsigargin (Thg). After treatment for 24 hours, cytosol (S100), ER, mitochondria (mito) and nuclei (Nuc) were biochemically fractionated from cells, and an aliquot of cells was used for total protein lysates (Total). Proteins in each fraction or total lysates were immunoblotted for MCC, Rhbdf1 (an ER protein), cIAP1, cIAP2, cyclin D2, COX IV (a mitochondrial protein), and actin. Results shown are representative of 3 independent experiments.