Fig. 1. Detection of the endogenous and ectopically expressed PCTAIRE-1 protein. A, protein extracts (15 µg) from various cell lines were examined by Western blotting using an antibody specific to PCTAIRE-1. Jurkat cells, Lane 1; COS-7 cells, Lane 2; Hs68 cells, Lane 3; NIH3T3 cells, Lane 4; human lung carcinoma A549 cells, Lane 5; human cervical carcinoma HeLa cells, Lane 6; human mammary carcinoma MCF7 cells, Lane 7; human gastric carcinoma GTL16 cells, Lane 8; human colon adenocarcinoma Colo 205 cells, Lane 9; 293 human embryo kidney cells, Lane 10; human epidermoid carcinoma A431 cells, Lane 11; lymph node prostate cancer LN-cap cells, Lane 12; human osteosarcoma Saos-2 cells, Lane 13; human glioblastoma U87 cells, Lane 14. B, extracts (10 µg) of wild-type (Lane 1) and SV40-transformed (Lane 2) WI38 fibroblasts were examined by Western blot analysis as described above. C, Western blot of GTL16 cell extract (15 µg). The membrane was probed with PCTAIRE-1 antibody (Lane 1) or with the same antibody preincubated with a 10-fold (w/w) excess of antigenic peptide (Lane 2). D and E, COS-7 cells ectopically expressing HA-tagged PCTAIRE-1 were extracted at 24 (Lane 3) or 36 h (Lane 4) after transfection. Controls include nontransfected cells (Lane 1) and cells transfected with empty vector (pcDNA3-HA; Lane 2). PCTAIRE-1 protein was revealed with the anti-PCTAIRE-1 antibody (D) or with the anti-HA 12CA5 antibody (E). The band at Mr 45,000 in E is nonspecifically detected by the antibody used to probe the membrane.