BAFF-R is present in different subcellular compartments in NHL-B cells. (A) RT-PCR analysis of BAFF-R mRNA expression in NHL-B-cell lines. (B) BAFF-R protein was stained for Cy3 (red) fluorescence and a nuclear marker TOPRO-3 (blue) in LBCL and MCL cell lines, and then analyzed by confocal microscopic analysis. (C) Western blot analysis (WB) of protein extract from different subcellular fractions (PM indicates plasma membrane; C, cytoplasm; NE, nuclear envelope; and NP, nucleoplasm) for BAFF-R, syndecan 4 (a plasma membrane marker), calreticulin (an endoplasmic reticulum marker), p62 (a nuclear envelope marker), and lamin B (a nuclear marker). (D) BAFF-R competition experiments were performed by preincubating a BAFF-R peptide with specific BAFF-R antibody before probing nuclear extracts from NHL-B cells. (E) Western blot analysis of 50 μg protein from cytoplasm and nucleoplasm for BAFF-R in normal peripheral blood B cells. Actin and lamin B were used as cytoplasmic and nucleoplasmic markers. (F) Confocal fluorescence microscopic images of GFP-BAFF-R fusion protein localization in a representative live NHL-B cell (MS), cotransfected with BAFF-R expression plasmid pcGFP-BAFF-R (green) and a nuclear localization expression plasmid pDsRed2-Nuc (red).