Fig. 2. ProAR is targeted to the nuclear envelope. (A) Schematic presentation of AR deletion mutants. All constructs (AR-V5, AR-C1-V5, AR-C2-V5, AR-C3-V5 and AR-C4-V5) were V5-tagged at the C-terminus. (B) Three monoclonal antibodies mAb2, mAb5 and mAb9 against the cytoplasmic domain of proAR were established. The core epitope region for each antibody is underlined. (C) Total lysates of HeLa cells expressing AR-V5 or AR-C-V5 mutants were analyzed by immunoblotting with the three mAbs. An anti-V5 mAb was used as a positive control. (D) Lysates of HeLa cells expressing wild-type AREG were immunoblotted with anti-AR-C mAbs. (E and F) HeLa cells transiently expressing wild-type AREG were incubated with or without TPA for 30 minutes and immunostained with the anti-AR-C mAbs (E) or mAb2 and anti-LAMP-1 mAbs (F). (G) HeLa cells transiently expressing wild-type AREG were treated with 50 nM Bafilomycin (Baf) for 60 minutes and incubated with TPA in the presence of Baf. The cells were then immunostained with mAb2 and anti-LAMP-1 mAb. Scale bars: 5 µm.