Figure 1. Characterization of AIbZIP in androgen-sensitive prostate cancer cell line LNCaP.

(a) Microarray datasets were accessed in the ONCOMINE Cancer Profiling Database (version 4.5, www.oncomine.org). The number of each tumor sample is described in Methods section. The y-axis represents the expression levels of normalized AIbZIP. The line within the box represents the median expression value for each group, and the upper and lower edges of the box indicate the 75% and 25% limits of distribution, respectively. The lines extending from each box (whiskers) indicate the 90% and 10% limits of distribution. (b) RT-PCR analysis for AIbZIP and AR in indicated cancer cell lines. GAPDH was used as an internal control. (c) RT-PCR analysis for AIbZIP in LNCaP cells treated with R1881 for indicated time periods. (d) Western blot (WB) analysis for endogenous AIbZIP in LNCaP cells treated with R1881 as in (c). Asterisk: nonspecific bands. β-actin was used as a loading control. (e) Schematic representation of the domain structures of human AIbZIP, OASIS, and ATF6. Amino acids colored in red indicate the S1P recognition site, the putative S2P recognition site, and the N-glycosylation site. (f) RT-PCR analysis for AIbZIP and ER stress markers in LNCaP cells treated with various kinds of ER stressors for 6 h, or R1881 for 24 h. uXBP1: unspliced forms of XBP1, sXBP1: spliced forms of XBP1, Tm: tunicamycin, Tg: thapsigargin, BFA: brefeldin A. (g) WB analysis for the processing of AIbZIP protein in LNCaP tet-off cells stably expressing FLAG-tagged AIbZIP treated with ER stressors or R1881 as in (f). (h) HeLa cells were transfected with a vector expressing FLAG-tagged AIbZIP for 24 h, then co-stained with anti-FLAG (AIbZIP) and anti-calnexin or anti-GM130 antibodies. Full-length gels and blots are presented in Supplementary Figure S8.