Double immunolabeling and immunoblot analyses of CagA expression in tumor cells and mucosa of gastric MALT lymphoma. (a) Confocal microscopy showing that most CagA-positive cells (green fluorescence) express CD20 (red fluorescence). (b) Double immunolabeling of CagA and CD20 in two H. pylori-dependent gastric MALT lymphoma cases with immunohistochemically identified CagA expression. Left: the majority of the tumor cells (red fluorescence, CD20) contain nuclear CagA (green fluorescence). Right: CagA labeling is present in the cytoplasm, whereas CD20 labeling is exclusively cytoplasmic. The red arrow in (b) represents CagA expression, CD20 expression, nucleus (DAPI), or the merged image of CagA and CD20 expression. The black arrow in (c) and (d) represents a molecular weight of approximately 120 kDa. (c) Lane 1: Western blot analysis identified one antigen, with a molecular weight of approximately 120 kDa, from the tumor cells of a H. pylori-dependent gastric MALT lymphoma case with CagA expression. Lane 2: An H. pylori-negative gastric MALT lymphoma case without CagA expression serving as a negative control. Lane 3: Pfeiffer cell (diffuse large B-cell) serving as a negative control (no CagA expression). Lane 4: Ramos cell (Epstein–Barr virus-negative Burkitt's lymphoma) serving as a negative control (no CagA expression). Lane 5: Raji (Epstein–Barr virus-positive Burkitt's lymphoma) serving as a negative control (no CagA expression). Lane 6: RL (an IgM-secreting low-grade lymphoma cell line) serving as a negative control (no CagA expression). (d) Lanes 1–4: Western blot analysis identified one antigen, with a molecular weight of approximately 120 kDa, from the tumor cells of 4 H. pylori-dependent gastric MALT lymphoma cases with CagA expression (immunohistochemical scores ranging from 15 to 65% staining intensity, moderate ++ to strong +++). Lanes 5 and 6: Western blot analysis indicated the absence of the CagA antigen in tumor cells of two H. pylori-independent gastric MALT lymphoma cases without CagA expression (immunohistochemical score 0).