Biological and regulatory properties of Vav-3, a new member of the Vav family of oncoproteins - PubMed (original) (raw)

FIG. 2

(A) Distribution of VAV-3 transcripts in adult human tissues. A filter containing 2 μg of poly(A) mRNA isolated from the indicated tissues (MTN blot; Clontech) was hybridized to either a VAV-3 (upper panel) or a ubiquitin (lower panel) probe as described in Materials and Methods. The hybridized filter was exposed for 48 h (upper panel) and 1 h (lower panel). The migration of the VAV-3 and ubiquitin mRNAs is indicated by arrows. The migration of molecular weight markers is indicated on the right. (B) Dot blot analysis of the expression of the VAV-3 gene in adult and embryonic human tissues. The hybridized filter (Human RNA Master blot; Clontech) contained poly(A) mRNA from the following sources: A1 (whole brain, 257 ng), A2 (amigdala, 135 ng), A3 (caudate nucleus, 161 ng), A4 (cerebellum, 109 ng), A5 (cerebral cortex, 99 ng), A6 (frontal lobe, 89 ng), A7 (hippocampus, 118 ng), A8 (medulla oblongata, 124 ng), B1 (occipital lobe, 207 ng), B2 (putamen, 202 ng), B3 (substantia nigra, 132 ng), B4 (temporal lobe, 117 ng), B5 (thalamus, 125 ng), B6 (subthalamic nucleus, 127 ng), B7 (spinal cord, 169 ng), C1 (heart, 410 ng), C2 (aorta, 106 ng), C3 (skeletal muscle, 210 ng), C4 (colon, 177 ng), C5 (bladder, 100 ng), C6 (uterus, 114 ng), C7 (prostate, 190 ng), C8 (stomach, 293 ng), D1 (testis, 213 ng), D2 (ovary, 205 ng), D3 (pancreas, 514 ng), D4 (pituitary gland, 331 ng), D5 (adrenal gland, 349 ng), D6 (thyroid gland, 169 ng), D7 (salivary gland, 393 ng), D8 (mammary gland, 93 ng), E1 (kidney, 213 ng), E2 (liver, 429 ng), E3 (small intestine, 275 ng), E4 (spleen, 175 ng), E5 (thymus, 209 ng), E6 (peripheral blood lymphocytes, 94 ng), E7 (lymph node, 164 ng), E8 (bone marrow, 147 ng), F1 (appendix, 152 ng), F2 (lung, 243 ng), F3 (trachea, 235 ng), F4 (placenta, 361 ng), G1 (fetal brain, 137 ng), G2 (fetal heart, 135 ng), G3 (fetal kidney, 210 ng), G4 (fetal liver, 243 ng), G5 (fetal spleen, 149 ng), G6 (fetal thymus, 104 ng), and G7 (fetal lung, 245 ng). The filter was exposed for 1 week. (C) Phosphorylation of Vav-3 in Jurkat cells. Cells were stimulated for the indicated periods of time, lysed, and immunoprecipitated with antibodies specific for Vav-3. Immunocomplexes were then subjected to anti-phosphotyrosine immunoblot analysis. The mobility of Vav-3 and the immunoglobulin G heavy chain is indicated by arrows. (D) Phosphorylation of Vav-3 proteins in COS-1 cells. Transfected cells were made quiescent and then stimulated for the indicated periods of time with EGF. Proteins were then immunoprecipitated with anti-GFP antibodies and immunoblotted with anti-PTyr antibodies (upper panels). The filter was then blotted with anti-GFP (middle panel) and, after being stripped with SDS at 50°C for 30 min, incubated with anti-Shc antibodies (lower panel). The position of the EGF-R, Vav-3, and Shc isoforms is indicated by arrows. Lanes 1 to 3 contain immunoprecipitated EGFP–Vav-3 (Δ1–144) (ΔN). Lane 4 contains immunoprecipitated EGFP–Vav-3 (Δ1–144+Δ607–847) (ΔN+ΔC). (C and D) The mobility of the coelectrophoresed molecular weight markers is indicated on the right of each panel. IP, immunoprecipitation; WB, Western blot.