CD28 signaling regulates Bim expression levels, and knockdown of Bim partially prevents CD28 blockade–induced apoptosis. (A) MM.1S cells were cultured for 72 hours in melphalan ± 10 μg/mL αCD28.2. Cells were lysed and RNA was collected. Semiquantitative RT-PCR was conducted (top) and assessed by densitometry (bottom). (B) MM.1S and U266 cells were cultured for 72 hours in serum-free conditions ± 10 μg/mL αCD28.2. Lysates were made and assessed by western blot. Densitometry was assessed using Quantity One software (bottom). (C) MM.1S cells were cultured for 72 hours in melphalan ± 100 μg/mL CTLA4-Ig. Lysates were made and assessed by western blot (top). Densitometry was assessed using Quantity One software (bottom). (D) U266 cells were cultured in full serum or serum-free media ± 100 μg/mL CTLA4-Ig for 48 hours. Lysates were prepared and Bim (left) or Mcl-1 (right) was immunoprecipitated and analyzed by western blot for Bim or Mcl-1 expression. Densitometry of all 3 Bim isoforms was averaged and compared relative to Mcl-1 in the Bim IP and was performed using Quantity One software (bottom). (E) MM.1S cells were transfected with Bim or scramble siRNA and Bim expression was assessed by western blot after 48 hours (top). Percent silencing was calculated using densitometry (bottom). (F) Cells transfected in panel E were plated in serum-free medium ± 100 µg/mL CTLA4-Ig. Cells were harvested after 48 hours and survival was assessed by Annexin V/7AAD staining on flow cytometry. Data are representative of 3 separate experiments except for panel D, which is representative of 2 separate experiments. **P < .01 ***P < .001.