Dependence of VEGF processing on ORP150 in hypoxic macrophages. (a and b) Macrophages were exposed to hypoxia in the presence of Ad/S-ORP150, Ad/AS-ORP150, AxCALacZ, or AxGFP. Cell lysates were subjected to Western blot analysis using either anti-ORP150 Ab (a) (upper panel) or anti-KDEL mAb’s (lower panels). RNA was also prepared from cells incubated in the presence of adenovirus. (b) RT-PCR analysis was performed using primers specific for either VEGF (upper panel), β-actin (middle panel), or GAPDH (lower panel). (c) Cell lysates were fractionated, as described, and fractions corresponding to ER (lanes 1 and 2), Golgi apparatus (lane 3), plasma membrane (lane 4), and cytosol (lane 5) were subjected to immunoblot analysis using cellular organelle-specific Ab’s. (d–i) Cultured macrophages were infected with AxCALacZ (100 moi), Ad/S-ORP150 (0–100 moi), or Ad/AS-ORP150 (0–100 moi), and further incubated under hypoxic conditions for 24 hours, either in the absence (d, e, g, and h) or presence (f and i) of SNP. The content of VEGF antigen in the ER fraction (d–f) and culture supernatant (panels g–i) was measured by ELISA as described (n = 6; mean ± SD). *P < 0.01 compared with AxCALacZ-treated culture by either multiple comparison analysis (d, g, h, and f) or multiple contrast analysis, following two-way ANOVA (i). (j–o) Macrophages were infected with either AxCALacZ (j–l) or Ad/AS-ORP150 (m–o) and immunostained with either anti-KDEL mAb (j and m) or anti-VEGF mAb. (l and o) Both images were digitally overlapped. ×400.