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Fig. 8. Characterization of antibody for ARAP2. (A) ARAP2 protein levels in cultured cells. ARAP2 was detected by western blotting using antibody 1185, which was raised against amino acid residues 1689-1704 of ARAP2 as described in Materials and Methods. HEK 293, human embryonic kidney 293 cells; U118, glioblastoma cells; U87, glioblastoma cells; Jurkat, T lymphocyte cells; MCF-7, breast adenocarcinoma cells; MDA-MB-231, advanced breast adenocarcinoma cells; LNCAP, prostate cancer cells. A lysate from cells transiently transfected with a plasmid directing expression of Flag-ARAP2 was used a positive control. Actin was used as a control for normalization. In the middle panel, the signal was blocked by incubating the antibody with the peptide (10 µg/ml) against which the antibody was raised. (B) Effect of siRNA treatment of cells on proteins detected by antiserum 1185. The indicated cell lines were treated for 72 hours with a irrelevant siRNA (control siRNA) or siRNA targeting the 3'UTR of ARAP2 and then lysed for immunoblotting. (C) Characterization of antiserum 1185 against ARAP2 in immunofluorescence. The effect of peptide treatment of antibody and siRNA treatment of cells was examined in the indicated panels. The cells were counterstained for paxillin. Note that the signal for paxillin is easily detected on siRNA treatment but is also altered by the siRNA as described in the article. (D) Antibodies affinity-purified from 1185 and 1187 antisera. Rabbit anti-ARAP2 antisera raised against the synthetic peptides RSRTLPKELQDEQILK, residues 1689-1704 of ARAP2 (antiserum 1185), and ANVHKTKKNDDPSKDY, residues 78-93 of ARAP2 (antiserum 1187) were affinity-purified and used for immunoblotting of lysates from MCF7. (E) Immunofluorescence with affinity purified antibodies. Immunofluorescence of MCF7 cells was performed as in (C) but with affinity-purifed antibodes from antisera 1185 and 1187.
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