Identification of the IRTA1 protein with specific antibodies. (A-B) The M-IRTA1 mAb. (A) A strong band of 51 kDa (arrow) of the expected molecular size for the external part of the IRTA1 protein is seen in the lane corresponding to lysates of IRTA1-HA-transfected COS cells but not in the control cells lane. (B) Identical results were observed following immunoprecipitation of the protein with M-IRTA1 and detection of Western blotted immunoprecipitates with anti-HA mAb; the arrow points to the IRTA1 band. (C-D) The anti-IRTA1 polyclonal antibody. (C) Northern blot (top) analysis of IRTA1 expression in transfectants and cell lines. Two specific RNA species (^*) are shown in the ER-EB cell line 24 and 48 hours after induction²  and in the CA46 cell line; the lower molecular weight bands represent the GAPDH (glyceraldehyde phosphate dehydrogenase) mRNA obtained by hybridization with the cognate probe in the same reaction (loading control). The bottom panel shows the Western blot analysis with the anti-IRTA1 purified polyclonal antibody on total cell extracts from vector- or full-length IRTA1-HA-transfected 293T cells and cell lines. Western blot with β-actin is shown as a control for protein loading. IRTA1 appears as 2 closely migrating bands of 70 to 75 kDa (black bars), which represent glycosylation isoforms (G.C., unpublished results, December 2002). (D) Colocalization of anti-IRTA1 polyclonal rabbit antibody (FITC, green) and anti-HA tag (TRITC, red) on transfected 293T cells. The bottom small panel shows DAPI staining on nuclei. Colocalization is seen as yellow signals in the superimposed image (large panel). Original magnifications: × 400 (large panel); × 4000 (small panels).