CD44 ligation stabilizes p27 protein. (A) NB4 cells (2 × 10⁵/mL) were treated at 37°C for 4 hours with 50 μM MG132 (a specific proteasome inhibitor³² ) or with DMSO or nothing (controls); cell extracts were carried out as indicated in “Materials and methods” and immunoprecipitated using a polyclonal rabbit anti-p27 mAb. Immunoprecipitates were processed for SDS-PAGE and Western blotting using polyclonal rabbit anti-p27 mAb as described in “Materials and methods.” Results represent 1 representative experiment of 2. (B) NB4 cells were treated with 2.5 μg/mL IgG1 (controls) or A3D8 for 24 hours and were pulsed-labeled for 1 hour with 200 μCi (7.4 × 10⁶ Bq)/mL [³⁵S]methionine (ICN). The cells were then chased with cold methionine for the indicated times. Cell lysates were immunoprecipitated with anti-p27 Abs. The p27 protein was detected after Western blotting by autoradiography and quantified using a PhosphoImager. Results represent 1 representative experiment of 2. (C) Cells were treated for 24 hours with A3D8, and then 15 mg/mL cycloheximide was added and p27 expression was analyzed 1 hour and 2 hours later by Western blotting. Immunoblots were quantified by densitometric scanning, and p27 protein levels (corrected with respect to α-tubulin expression) were expressed relative to values obtained before cycloheximide treatment (arbitrarily 100%). Results represent 1 representative experiment of 3.