ABSTRACT
Figure 8 Immunoprecipitation of Rll. HL60 cells were grown in serum-free medium with and without 100 nM [3H]RA. Cell extracts were labeled by 8-azido-e2P]cAMP and immunoprecipitated with anti-RII antiserum. Lane 1 is a portion from the solubilized fraction, untreated with antiserum and protein A-Sepharose. Lane 2 shows the immunoprecipitate from the initial solubilized fraction carried out with protein A but without antiserum. Lanes 3 and 4 show the immunoprecipitated 8-azido-e2P]cAMP labeled protein (lane 4) and the nonimmunoprecipitated proteins remaining in the soluble fraction (lane 3) after treatment of the solubilized fraction with 8 111 of anti-RII antiserum and protein A-Sepharose. The gel (lanes 1-4) was exposed to the film for 3 days. Lanes 5-7 show the levels of protein specifically immunoprecipitated as a function of increasing concentrations of anti-RTI antiserum (lane 5, 4~tl; lane 6, 8!11; and lane 7, 16~tl). The gel (lanes 5-7) was exposed to the film for 13 days. Lanes 8-11 show the specificity of this immunoprecipitation with the anti-RII antiserum. Lane 8, 4 111 of anti-RII antiserum; lanes 9-11, 4, 8, and 16 111 of normal rabbit serum, respectively. The gel (lanes 8-11) was exposed to the film for 13 days. The cells used for lanes 1-4 were grown with 100 nM eH]RA. The cells used for lanes 5-11 were grown without RA. The band containing the radioactivity shown in lane 4 was cut from the gel and the 32P and 3H determined in a liquid scintillation counter. We found 1.2 frnol of RA and 0.4 fmol of 8-azido-cAMP present in this band.
