An immunoaffinity purification procedure for sv40 large t antigen

Characterization of the Serological Response in Man to the Latent Membrane Protein and the Six Nuclear Antigens Encoded by Epstein-Barr Virus

“…The spent cell lysate was immediately used for immunoprecipitation of LMP, in a manner similar to that described for EBNA 5 except that the LMP antibodies (CS. 1 to 4; Rowe et al, 1987 a) were first covalently immobilized to Protein A-Sepharose beads, to which rabbit anti-mouse antibodies had been pre-adsorbed, according to the method of Simianis & Lane (1985). In this way, the contamination of the LMP preparation by both rabbit and mouse immunoglobulins was minimized.…”

SV40 large T antigen binds to the TBP-TAF(I) complex SL1 and coactivates ribosomal RNA transcription.

“…As a first step, rRNA was transcribed using HeLa cell nuclear extract in the absence or presence of recombinant large T antigen purified from Sf9 insect cells. Large T antigen protein was purified to apparent homogeneity using either conventional chromatography or antibody-affinity chromatography (Clark et al 1983;Simanis and Lane 1985). As shown in Figure 2A, addition of purified T antigen to a HeLa cell nuclear extract resulted in an increase in the level of rRNA synthesis (lanes 2,3).…”

PAK1 and aPKCζ Regulate Myosin II-B Phosphorylation: A Novel Signaling Pathway Regulating Filament Assembly

“…Cdk7 antibodies or beads only were used as negative control for the coimmunoprecipitation experiments. The covalent coupling was performed essentially as described (Simanis and Lane, 1985). Briefly, the beads were washed twice with 0.2 M sodium borate, pH 9.0, incubated o/n at RT with 20 mM dimethyl pimelimidate dihydrochloride and then washed twice and incubated for 2 h at RT with 0.2 M ethanolamine, pH 8.0.…”