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. 2015 Dec 1;128(23):4353–4365. doi: 10.1242/jcs.176057

Fig. 1.

Fig. 1.

Luman is induced during osteoclast differentiation and cleaved in response to RANKL signaling. (A) Schematic representation of the domain structure of murine Luman, BBF2H7, OASIS and ATF6α. The basic leucine zipper (bZIP; basic region and leucine zipper), putative transmembrane domain and luminal domain are indicated. (B) Western blot analysis for Luman protein in cell lines originally derived from bone tissues: ATDC5, chondrocytes; MC3T3-E1, osteoblast-like cells; and RAW264, macrophages. Cells were cultured with or without 1 µM MG132 proteasome inhibitor for 6 h. Cell lysates were separated with SDS-PAGE, and western blotting was performed with an antibody against Luman. (C) RAW264 cells were cultured with 1 µM brefeldin A (BFA), 1 µg/ml tunicamycin (Tm), or 1 µM thapsigargin (Tg) for the indicated time periods. Western blotting was performed with an antibody against Luman (upper panel). Middle panel shows the expression levels of actin used as the internal control. Lower panels show RT-PCR analysis of XBP-1 and GAPDH mRNA in each sample. Note that spliced forms of XBP-1 (XBP-1-s) were detected in cells that had been treated with tunicamycin and thapsigargin, but Luman N-termini were not detected under such conditions. (D) RAW264 cells were cultured with RANKL for the indicated time periods. Cell lysates were subjected to western blotting with an antibody against Luman. Relative quantified data of Luman N-terminus bands are indicated below the Luman blot. The intensity of the Luman N-terminus band in 0 h without MG132 treatment was set as 1.0. Arrowhead indicates Luman N-terminus bands. (E) BMMs were cultured with M-CSF and RANKL for the indicated time periods. Cell lysates were analyzed by western blotting with an antibody against Luman. The mRNA expression of the indicated genes was determined by using RT-PCR analysis. (F) BMMs were cultured with M-CSF and RANKL in the presence of 1 µM MG132 for 6 h. Cell lysates were analyzed by western blotting with an antibody against Luman. Note that the Luman N-terminus was detected in the presence of MG132 (arrowhead). For C–F, cell lysates from Luman-transfected HeLa cells were used as a positive control. Asterisk indicates non-specific bands.