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Fig. S5 Depletion of H2B-GFP by Ab-SPOP E3 ligases engineered with alternative GFP nanobodies. 293TetOn cells expressing H2B-GFP were transfected with vectors expressing four different Ab- SPOP ligases (GBP1-SPOP, GBP2-SPOP, GBP6-SPOP, and GBP7-SPOP) from the bi-directional TRE promoter. Expression of TagRFP and depletion of H2B-GFP were measured 8 hours after adding doxycycline (1 µg/ml) to media. ‘–‘ indicates untransfected cells. Blue signal in the merged Hoechst/H2B-GFP panel indicates cells with depleted nuclear H2B-GFP. All cells expressing TagRFP had depleted H2B-GFP, however some cells with depleted H2B-GFP did not express TagRFP, consistent with the observation that nuclear depletion begins 3 hours after activation of the TRE promoter (Supplementary Figs. 3 and 6).