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Fig. 1. Affinity purified anti-UAP56 and -URH49 antibodies are highly specific. (A) HeLa and CaSki cell extracts were separated on 10% SDS-PAGE gels probed with unfractionated peptide antibodies generated against UAP56 in rabbit. (B) Affinity purified antibodies detected GST-UAP56 but not GST-URH49. Bacterial lysates containing either GST-UAP56 or GST-URH49 were separated by SDS-PAGE and probed with unfractionated antiserum, which detected both proteins. Affinity purified anti-UAP56 antibody was prepared by passing the antiserum through a GST-URH49 affinity column to remove cross-reacting antibodies. The eluate was then passed through a GST-UAP56 affinity column. Bound antibodies were eluted with glycine HCl, pH 2.0. After affinity purification, the anti-UAP56 antibody detected only GST-UAP56 but not GST-URH49. (C) Affinity purified antibodies against GST-UAP56 and GST-URH49 show that both proteins are located at RNA-splicing speckled domains. HeLa cells were permeabilized, fixed and stained with affinity purified antibodies. (D) HeLa cells were treated with a pool of four siRNA oligonucleotides against UAP56. After 76 hours of treatment, the cells were fixed and immunostained with affinity purified antibodies against UAP56. This treatment removed all detectable UAP56 within the nucleus, demonstrating the high specificity of affinity purified antibody. Both micrographs were collected at the same microscope settings. They are presented as moderately overexposed to show that about 10% of UAP56 remains even after knockdown. Scale bars: 10 µm. (E) The affinity purified anti-UAP56 antibody was used to probe a western blot of protein extracts either from MCF-10A cells treated with a UAP56-specific shRNA knockdown for 76 hours or from control cells. The antibody signal was greatly reduced in the knockdown cells.
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