![Fig. 7. Expression of HA-p21 in transduced cells. / (A) Western blot analysis with the anti-HA mAb. UT-7 cells (lane 1) grown with GM-CSF were infected at day 2 of culture with VSV-G–pseudotyped retrovirus containing an EGFP vector alone (lane 2) or an HA-tagged p21 and EGFP (lane 3). CD34+cells, isolated from cytapheresis samples (lane 4) cultured with PEG-rHuMGDF and SCF and were infected with the same supernatants (containing HA-tagged p21 [lane 6] or EGFP alone [lane 5] at days 5 and 6. Murine lin− cells cultured for 2 days with PEG-rHuMGDF and SCF (lane 7) were infected with an ecotropic retrovirus containing the same HA-tagged p21 and EGFP (lane 8). Similarly, lin− marrow cells from p21−/− mice were infected with the ecotropic retrovirus coding for the HA-tagged p21 and EGFP (lane 9). Protein lysates were obtained 48 hours after infection and blotted with the anti-HA mAb. Western blots are from a representative experiment (n = 2). (B) Human CD34+ cells and UT-7 cells were infected with the VSV-G–pseudotyped retrovirus containing the HA-tagged p21 vector (identical to lanes 3 and 6 of panel A). Protein lysates were obtained 48 hours after infection and blotted with an anti-p21 mAb. The wild-type p21 and the HA-tagged p21 could be detected. Overexpression of p21 was about 4-fold in normal megakaryocytes and more than 15-fold in the UT-7 cells when the ratio between the tagged p21 and the wild-type p21 was studied. Western blots are from a representative experiment (n = 2).](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/98/12/10.1182_blood.v98.12.3274/6/h82311837007.jpeg?Expires=1735502117&Signature=4U0k8ACPfsVZWE~HpwCKFo2~qKQsSFTa6i7yPo6Mmy0nBj5r5RAYDNV8iB5M-BimICjvSnm3eY4qgRBjWHx~-TmATVHCaol6aqS0GzbzW3ERsm5tAM-kp3Ttn5cz1ADPLpY4TITEBtJ77bXi72m-nhuK4Efqy~reU18XhGHJ4N5Na3TAxBdq9OSWlYfNOQoKVPsVYyxGg2AbgP0OOoahu3hunU-BtK7nB08ZSUUVDOHewwTVRdCLnqH1hoTGkZYGs8v2D9OSTfYrIeR4E6xOAEsv9hFR7oCo4wrFyLPIX7gO5wL0h46WYVUXvd1w57lcHi3yn3ih3m~mEBl12m~yXA__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Expression of HA-p21 in transduced cells.
(A) Western blot analysis with the anti-HA mAb. UT-7 cells (lane 1) grown with GM-CSF were infected at day 2 of culture with VSV-G–pseudotyped retrovirus containing an EGFP vector alone (lane 2) or an HA-tagged p21 and EGFP (lane 3). CD34+cells, isolated from cytapheresis samples (lane 4) cultured with PEG-rHuMGDF and SCF and were infected with the same supernatants (containing HA-tagged p21 [lane 6] or EGFP alone [lane 5] at days 5 and 6. Murine lin− cells cultured for 2 days with PEG-rHuMGDF and SCF (lane 7) were infected with an ecotropic retrovirus containing the same HA-tagged p21 and EGFP (lane 8). Similarly, lin− marrow cells from p21−/− mice were infected with the ecotropic retrovirus coding for the HA-tagged p21 and EGFP (lane 9). Protein lysates were obtained 48 hours after infection and blotted with the anti-HA mAb. Western blots are from a representative experiment (n = 2). (B) Human CD34+ cells and UT-7 cells were infected with the VSV-G–pseudotyped retrovirus containing the HA-tagged p21 vector (identical to lanes 3 and 6 of panel A). Protein lysates were obtained 48 hours after infection and blotted with an anti-p21 mAb. The wild-type p21 and the HA-tagged p21 could be detected. Overexpression of p21 was about 4-fold in normal megakaryocytes and more than 15-fold in the UT-7 cells when the ratio between the tagged p21 and the wild-type p21 was studied. Western blots are from a representative experiment (n = 2).
