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. 2020 Dec 28;7(2):274–291. doi: 10.1021/acscentsci.0c01337

Figure 3.

Figure 3

Robust RAS degradation with doxycycline-inducible anti-RAS biodegraders. (a) Western blot analysis of T-REx-293 cells with stable integration of K27-SPOP (or its controls) under the control of a Tet-responsive promoter. Various concentrations of doxycycline (1 or 10 ng/mL) were added to the culture media for the indicated length of time (4 or 24 h), and protein lysates were collected. Degradation of RAS was detected using a pan-RAS antibody, and disruption to the MAPK pathway was measured using the levels of phospho-ERK1/2. Expression of K27-SPOP (or its controls) was detected using an anti-FLAG-tag antibody. HSP90 was used as a loading control. (b) Incucyte confluency measurements of T-REx-293 cells with stable integration of K27-SPOP (or its controls) under the control of a Tet-responsive promoter. Various concentrations of doxycycline (0.1 to 100 ng/mL) were added to the culture media, and the percentage confluency of the cells was tracked continuously over 4 days. (c) Western blot analysis as in (a) on protein lysates collected at 1, 2, or 4 days after treatment with 1 ng/mL doxycycline.