2D-PAGE and mass spectrometry identify the 28-kDa protein as TPD52. (A-B) Spots S1 and S2 (corresponding to the 28-kDa band in 1D-Western blots in Figure 1) are shown in a silver-stained 2D gel of B28p-positive lymphoma cells (A) and in the corresponding 2D-Western blot probed with the B28 mAb (B). Spot S3, which represented an unspecific reaction also detected in the negative control (U937 cell line; not shown), was not subjected to mass spectrometry analysis. (C) Peptide spectra. Protein S2 was isolated from a Coomassie-stained 2D gel and digested with trypsin. The mass of the resulting fragments was assessed by MALDI-TOF spectrometry. Peptide masses corresponding to TPD52 in S2 are denoted with asterisks: 1222.644 Da, 1454 Da, 1657 Da, and 2099 Da. (D-E) B28p strongly stains Phoenix cells transfected with a construct encoding human TPD52 (D). In contrast, no reactivity is seen toward cells transfected with any of the constructs encoding TPD52L2 (E) or TPD52L1, murine Tpd52, and the empty vector (not shown). The APAAP technique was used with hematoxylin counterstain. Original magnification, × 1000.