Figure 3.

Expression of p21 and p24 in MEFs. (a) Fibroblast cultures were prepared from 12- to 13-day-old embryos from homozygous H-2k^btsA58 males × normal females (TMEFs) and normal males × normal females (NMEFs). The H-2K^btsA58 mice harbor the tsA58 (41) early region coding sequences under the control of the mouse major histocompatibility complex H-2K^b class 1 promoter (29). Expression from this promoter can be enhanced by exposure of the cells to γ-IFN. Fibroblasts derived from these mice are dependent upon the presence of γ-IFN as well as the permissive temperature for growth (17, 29). Embryo fibroblasts were prepared and grown in medium containing 10 units/ml γ-IFN where indicated (17). Cells were plated at a density of 1 × 10⁶ cells per 10-cm dish or 2.7 × 10⁶ cells per 15-cm dish. Cultures were serially cultivated every 4 days using this passaging regime until the cell number obtained after growth for 4 days was the same as the number of cells plated. At this stage the cells had ceased dividing and were considered to have become senescent. Total cell extracts were prepared 2 days after plating and analyzed for p24 and p21 by immunoblotting using 30 μg protein. (b) TMEFs were serially cultivated at 33°C plus γ-IFN or 39.5°C in the absence of γ-IFN. NMEFs were passaged at 39.5°C without γ-IFN. Total cell extracts were prepared and analyzed for p24 by immunoblotting. (c) Extracts were prepared from passage 12 TMEFs cultured at either 33°C plus γ-IFN or after shift up to 39.5°C for 3 days. Total protein (30 μg) was analyzed for p21 and p24 by immunoblotting with antibodies SX118 and 471. SX118 is another mouse mAb specific for p21 (provided by X. Liu, Ludwig Institute for Cancer Research, London). (d) Expression of p21 and p24 was analyzed in extracts prepared from p21−/− MEFs (30) as well as TMEFs and NMEFs. Total protein (50 μg) was analyzed initially with antibody 471 to detect p24 and then reprobed with antibody SX118 to detect p21.