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. 2005 Sep 1;102(37):13212–13217. doi: 10.1073/pnas.0506306102

Fig. 3.

Fig. 3.

Kinetics and dose–responsiveness of Rb regulation by pPRIME–TET–GFP–Rb. (A) Clonal isolates of the HeLa Tet-OFF cells expressing pPRIME–Tet–GFP–Rb (see Fig. 2H) were grown under inducing conditions (-DOX) and immunoblotted for Rb, GAPDH, and GFP protein levels. (B) Tet-OFF clone 1 (see A) expressing GFP–miR30–Rb was cultured for 1 week in medium containing the indicated concentration of DOX, and cell extracts were blotted for the indicated proteins. (C) Tet-OFF clone 1 was cultured for 1 week in the presence of 1 μg/ml DOX (repressing condition). At day 0, the drug was withdrawn from the culture medium, and cells were harvested at the indicated time points. Whole-cell extracts were analyzed by Western blotting for the indicated proteins. (D) Tet-OFF clone 1 was cultured for 1 week under inducing conditions (-DOX), followed by addition of DOX at a concentration of 1 μg/ml. Cells were harvested just before DOX treatment (day 0) and at the indicated time points. Whole-cell extracts were analyzed by Western blotting for the indicated proteins.