Sample preparation for microscopy

HeLa (ATCC), U2OS, and MEF cells were cultured in DMEM (Gibco) supplemented with 10% (v/v) FBS and 100 U/mL penicillin and 100 ug/mL streptomycin (Hyclone). RPE1 (ATCC) cells were cultured in DMEM/Ham’s F-12, 50/50 mix (Corning). The U2OS and MEF cells used were previously described⁵³. Cells were incubated for 1 hour with 1 μM bortezomib (Ubiquitin-Proteasome Biotechnologies), 10 μM E1 inhibitor (Compound 1; provided by Takeda Oncology, Cambridge, MA) or vehicle alone (0.1% DMSO). Each experiment was performed a minimum of two times.

We fixed cells at <80% confluence with 4% paraformaldehyde in PBS for 30 min at 37 °C, permeabilized them with 0.1% Triton X-100 for 10 min at room temperature and blocked for 1 hour with 5% BSA and 0.5% Tween 20 in PBS. Cells were stained for free Ub with 100 nM tUI-HA diluted in blocking solution for 30 min at room temperature. As a negative control, the sensor was pre-incubated for 5 min at room temperature with 100 μM Ub in blocking solution before addition to the samples. Next, cells were incubated overnight at 4 °C with anti-HA antibody (Sigma-Aldrich clone HA-7 or Bethyl Laboratories A190–108A; 1:1000 dilution), stained with Alexa Fluor 568-conjugated goat anti-mouse IgG (Thermo Fisher; 1:500 dilution), and mounted on slides using ProLong Diamond Antifade medium (Thermo Fisher). Some coverslips were also stained with anti-Ub (clone FK2, Enzo Life Sciences; 1:1,000 dilution) or anti-K48Ub (clone Apu2, Millipore; 1:200 dilution) primary antibodies and, subsequently, with Alexa Fluor 568-conjugated goat anti-mouse and Alexa Fluor 488-conjugated goat anti-rabbit (Thermo Fisher; 1:400 dilution) secondary antibodies. The HCS CellMask dye (Thermo Fisher) was added to the cells for 0.5 h as a marker of cell boundaries for high-content fluorescence intensity-based measurements.