FIGURE 2.

RIAM is required for melanoma cell invasion. A, immunohistochemical analysis of RIAM expression on melanoma lymph node metastasis. Sections were stained with the melanoma-specific marker HMB-45 (tumor) or with anti-RIAM antibodies or with DAPI to visualize nuclei. Sample 82 is shown. Additional analyses of melanoma specimens are shown in supplemental Fig. S1. B, lysates from BLM cells exposed for 15 min to CXCL12 were incubated with GST or GST-Ral-GDS, and bound Rap1 and RIAM were detected by immunoblotting. C, expression of RIAM in untransfected BLM cells, and in Mock and stable RIAM knockdown transfectants (D11 and F7), was analyzed by immunoblotting with anti-RIAM antibodies (top panel). Numbers below the gel represent densitometer analyses in arbitrary units. Bottom panel, transfectants were tested in invasion assays across Matrigel. D, BLM cells were transfected with control or with the indicated RIAM siRNA, and transfectants were analyzed by immunoblotting (top panel) or subjected to invasion assays (bottom panel). E, BLM cells were transfected with His- or Myc-tagged RIAM vectors, and transfectants were analyzed by immunoblotting to determine RIAM-His or RIAM-myc expression (left panel) or subjected to invasion assays (right panel). F, mock and D11 cells were transfected with control or RIAM-His vectors, and their invasion to CXCL12 was compared with that of mock cells. G, BLM cells were transfected with the indicated siRNA together with Rap1 CA, RIAM-His, or mock vectors. Transfectants were analyzed by immunoblotting (top panel) or tested in invasion assays (bottom panel). (C, n = 5; D, n = 3; E, n = 3; F, n = 2; G, n = 2.) ***, invasion was significantly inhibited, p < 0.001; **, p < 0.01; ^ΔΔΔ, invasion was up-regulated compared with invasion by mock transfectants, p < 0.001; ^ΔΔ, p < 0.01; ^Δ, p < 0.05. Anti-β-actin antibodies were used to control protein loading in Western blots.