![Fig. 4. Effect of rapamycin on the expression of proteins regulating early G1 progression in activated B-CLL cells. / Purified B-CLL cells were cultivated for up to 72 hours in the presence of DSP30 and IL-2. Rapamycin (Rap) was added where indicated at 50 ng/mL. (A) To quantify DNA synthesis, [3H]-thymidine was added for 8 hours at the indicated times (24, 48, or 72 hours), and 3H incorporation was measured. p27 was revealed by immunoblotting with mAb in total cell lysates. (B) The same membrane was sequentially stripped and probed with anti–cyclin D2, anti–cyclin D3, and anti-cdk4. (C) Hypophosphorylated (pRB) and hyperphosphorylated RBs (ppRB) were detected with the G3-245 mAb. (D) Tonsillar B cells were stimulated with DSP30 and IL-2 and cultured with or without rapamycin and analyzed for p27 expression. Protein concentrations were normalized by the Bio-Rad assay method. To control for equal protein loading, amido black staining was performed (data not shown).](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/101/1/10.1182_blood-2002-01-0189/6/h80133615004.jpeg?Expires=1735577085&Signature=VXjWQxfXCtLatqoSt5uQbYKQYhr3DjNRjQFI7B3FWici~sF0sM~WJuF9pIMzZLqXPbhUsSvNdqYpNKTGrbhyVyNfUuibeYOTXiZkaoyhxEpTyxGHa63BPQkip3-IPr22NR2WRD7duoMDGgDMq2K4zhEx8ie4Y2iZI4FQrNjO4OghXw2FWu5aQfZTJrML765NyIq-Ct9EB-07nFOfAe9FnCt24dhr0BW~VnoULeoGwHFF6H7fabFEZWMyvC7pL~I9AIrrgxq0ki2f~OMCq4wppt~kCH83U39ogY5mYWipj4Uq0T~AGhy3qVEOPUPA~2C~gh8sMbLl~ZWpQihjgWn5Fw__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Effect of rapamycin on the expression of proteins regulating early G1 progression in activated B-CLL cells.
Purified B-CLL cells were cultivated for up to 72 hours in the presence of DSP30 and IL-2. Rapamycin (Rap) was added where indicated at 50 ng/mL. (A) To quantify DNA synthesis, [3H]-thymidine was added for 8 hours at the indicated times (24, 48, or 72 hours), and 3H incorporation was measured. p27 was revealed by immunoblotting with mAb in total cell lysates. (B) The same membrane was sequentially stripped and probed with anti–cyclin D2, anti–cyclin D3, and anti-cdk4. (C) Hypophosphorylated (pRB) and hyperphosphorylated RBs (ppRB) were detected with the G3-245 mAb. (D) Tonsillar B cells were stimulated with DSP30 and IL-2 and cultured with or without rapamycin and analyzed for p27 expression. Protein concentrations were normalized by the Bio-Rad assay method. To control for equal protein loading, amido black staining was performed (data not shown).
