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. 1998 Aug 3;188(3):561–575. doi: 10.1084/jem.188.3.561

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70% of pp29/30 associates with the TCR–CD3–ζ complex in PBLCPCS cells. Digitonin lysates corresponding to 3 × 10⁷ PBLCPCS cells were consecutively incubated five times with 50 μl of packed CNBr-Sepharose beads coupled with purified OKT-3. (A) Lysates corresponding to 5 × 10⁵ cells before the first (no precl.) and after the fifth (5x precl.) preclearing step were separated by reducing 14% SDS-PAGE and analyzed by anti-pp29/30 and anti–MAP kinase Western blotting. The densitometric analysis of the individual lanes is indicated. (B) Immunoprecipitates corresponding to the first (no precl.), the third (3x precl.), and the fifth (5x precl.) preclearing were blotted onto nitrocellulose and analyzed for coprecipitation of TRIM and ζ chains by Western blotting. The protein bands migrating at 25 and 50 kD (right) represent the heavy and light chains of the precipitating mouse mAb.

70% of pp29/30 associates with the TCR–CD3–ζ complex in PBLCPCS cells. Digitonin lysates corresponding to 3 × 10⁷ PBLCPCS cells were consecutively incubated five times with 50 μl of packed CNBr-Sepharose beads coupled with purified OKT-3. (A) Lysates corresponding to 5 × 10⁵ cells before the first (no precl.) and after the fifth (5x precl.) preclearing step were separated by reducing 14% SDS-PAGE and analyzed by anti-pp29/30 and anti–MAP kinase Western blotting. The densitometric analysis of the individual lanes is indicated. (B) Immunoprecipitates corresponding to the first (no precl.), the third (3x precl.), and the fifth (5x precl.) preclearing were blotted onto nitrocellulose and analyzed for coprecipitation of TRIM and ζ chains by Western blotting. The protein bands migrating at 25 and 50 kD (right) represent the heavy and light chains of the precipitating mouse mAb.