Fig. 1.

Dual-input regulation of exogenous protein expression. a–d Dual-input regulation system consisting of the reverse transactivator (rtTA) and a stable (a) or conditionally destabilised (c) mCherry fluorescent protein. mRNA (b, d, left panels) and protein levels (b, d, right panels) measured in EF1a-rtTA_TRE3G-mCherry (b) and EF1a-rtTA_TRE3G-DDmCherry (d) mESCs treated for 24 h with Doxy (1000 ng/mL) or Doxy/TMP (1000 ng/mL and 100 nM, respectively). e Experimental scheme of protein half-life measurement. Following 14 h of treatment with Doxy (1000 ng/mL) or Doxy/TMP (1000 ng/mL and 100 nM, respectively), EF1a-rtTA_TRE3G-mCherry and EF1a-rtTA_TRE3G-DDmCherry mESCs were cultured in presence of the protein synthesis inhibitor cycloheximide (CHX, 25 μg/mL) and combination of Doxy (1000 ng/mL), TMP (100 nM) and Doxy/TMP. Protein half-life was measured by western blot after the indicated times of treatment. f, g Western blot densitometric quantification of EF1a-rtTA_TRE3G-mCherry (f) and EF1a-rtTA_TRE3G-DDmCherry (g) mESCs. Plotted mCherry and DDmCherry values are normalised against the housekeeping gene GAPDH. Data are means ± SEM (n = 2, b and d; n = 3, f and g). p values from two-tailed unpaired t test (b, d) and nonparametric one-way ANOVA (f, g) are shown. Source data are provided as a Source Data file