Protein samples were analyzed by standard polyacrylamide gel electrophoresis (PAGE) techniques. For preferential detection of high-molecular weight SUMO conjugates, PAGE was performed with lysates followed by wet transfer (100 V for 1 h) in a buffer consisting of 0.1% SDS, 10% methanol, 50 mM Tris-HCl, and 380 mM glycine. For preferential detection of free SUMO and lower molecular-weight SUMO conjugates, TCA extracts were analyzed by PAGE, followed by semi-dry transfer using a Power Blotter system (Thermo Fisher). Chemiluminescence-based imaging was performed using the MicroChemi imager (DNR). For quantification of signals, TIFF-format images were analyzed using ImageJ software (version 1.52a; NIH). Specifically for determining sumoylation levels, all SUMO signals (above the Ubc9-SUMO band, if it was present) were considered SUMO conjugates, and normalization was made to the corresponding signal from GAPDH immunoblots. Antibodies used for immunoblot analyses were: 1:500–1:1000 SUMO/Smt3 (y-84; Santa Cruz, sc-28649); 1:3000 GAPDH (Sigma, G9545); 1:500 Ubc9 (Santa Cruz, sc-6721); 1:3000 histone H3 (Abcam, ab1791); 1:1000 Myc epitope tag (Sigma, 05-724); 1:1000 Rpb3 (Abcam, ab202893); 1:5000 HA (12CA5; Sigma, 11583816001). The RPL3 antibody, which we used at a dilution of 1:200, was deposited to the DSHB by Warner, J. R. (DSHB Hybridoma Product ScRPL3 supernatant).