FIGURE 3.

Ubiquitination of SNAP-Tac proteins. HeLa cells were transfected with the indicated SNAP-Tac construct together with HA-ubiquitin and MARCH8-FLAG where indicated. Cells were labeled with BG-PEG₁₂-biotin for 1 h at 37 °C and then harvested for denaturing pulldown with NeutrAvidin-agarose and subsequent immunolabeling. A, immunoblot from pulldown with anti-HA and detection with DyLight 800 goat anti-mouse secondary antibody. B, immunoblot (IB) pulldown with anti-SNAP and detection with Alexa Fluor 680 goat anti-rabbit secondary antibody. C, merged image. D, immunoblot of MARCH8 expression from the cell lysate ($\raisebox{1ex}{$1$}\!\left/ \!\raisebox{-1ex}{$75$}\right.$ of the total volume) with anti-FLAG. The migration of prestained molecular mass markers (in kDa) is shown to the left of each panel.