Figure 4.

Detection of changes in SUMO conjugate states by cellular stress inducers. (a) U2OS cells treated with siRNA targeting endogenous SUMO1 or SUMO2/3 and doxycycline-treated to induce siRNA-resistant FLAG-SUMO1 or siRNA-resistant FLAG-SUMO2 for 48 h before treatment with indicated stress agents. UT (Untreated control), IR (Ionising Radiation, 4 Gy, 1 h post), CPT (Camptothecin 1 µM, 3 h), HU (Hydroxyurea 5 mM, 3 h), H₂O₂ (Hydrogen Peroxide 125 µM, 15 min), LPS (Lipopolysaccharide 1000 ng, 3 h), MG132 (5 µg/mL, 3 h), CB-5083 (0.1 µM, 3 h), 17-AAG (5 µM, 3 h), Sorbitol (500 mM, 30 min), heat shock (42 °C, 10 min), ICRF-193 (100 µM, 1 h). Immunoblots were probed with anti-FLAG antibody. Exposure time is shown, all exposures are shown in Supplemental Fig. 4a,b. (b) Representative immunoblots of lysates (a) probed with MAbs raised against SUMO1 in red (21C7, 4D12, BD8B16, D11, EP298 and Y299) and SUMO2/3 in blue (18H8, 3H12, 2H8, 852908, 1E7 and EPR4602) with exposure times shown. All exposures and Mabs tested are shown in Supplemental Fig. 4a,b. (c) Heat map relative quantification summarising changes in signal after treatment for each SUMO1 or SUMO2/3 MAb. % Change calculated from three experimental repeats per MAb using densitometry of proteins migrating above 70 kDa as a % change relative to the untreated control lane for each immunoblot. Free SUMO was not included in the quantification. The mean for each condition and MAb is shown. Red indicates a reduction in SUMO conjugates relative to siNTC, white no change and blue an increase. (d) U2OS cells treated with siRNA targeting endogenous SUMO4 and expressing siRNA-resistant FLAG-HA SUMO4 and treated as for (a). Lysates were then probed with MAbs raised to SUMO4 (orange). All exposures are shown in Supplemental Fig. 4a–c.