Sc 9060

Cells were lysed in a buffer containing 2% SDS and 67 mM Tris-HCl, pH 6.8, before being sonicated for 20 seconds twice. The same amount (30 μg) of protein (measured by the Lowry method) from each cell lysate were resolved by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred on to Immobilon-P membranes for 2 hours at 60 V. The membranes were incubated with the following antibodies: anti-p21Waf-1 mouse monoclonal antibody (diluted 1:200; Ab-1, OP64, Calbiochem); anti-UBC9 (UBE2I) rabbit polyclonal antibody (diluted 1:300; ab-30505, Abcam); anti-SENP2 rabbit polyclonal antibody (diluted 1:3,000; made by immunizing a rabbit with the antigen containing the amimoacids 111–358 of human SENP2); anti-SUMO-1 (FL-101) rabbit polyclonal antibody (sc-9060, Santa Cruz Biotechnology); and anti-actin (Clone C4) mouse monoclonal antibody (diluted 1:5,000; 691001, MP Biomedicals). After incubation with the appropriate peroxidase-coupled secondary antibody (diluted 1:2,500; Bio-Rad), immunocomplexes were detected by enhanced chemiluminescence (ECL) (Biological Industries).

Normal colon and CRC cell lines were fixed and permeabilized with ice-cold 100% methanol for at least 15 min at −20°C. Cells were blocked for 30 min in blocking buffer (0.25% BSA, 0.5% gelatin) and then incubated overnight with the following primary antibodies: rabbit anti-TET1 (1:500; Genetex GTX124207), rabbit anti-5hmC (1:500; Abcam ab214728 [RM236]), mouse anti-PML (1:500; Abcam ab96051), mouse anti-coilin (1:500; Abcam ab87913), SUMO1 (1:500; Santa Cruz Sc9060), and SUMO2/3 (1:500; Santa Cruz Sc32873). Cells were then incubated for 1 h with fluorochrome-labelled goat secondary antibodies: anti-rabbit Alexa Fluor 488 (1:500; Abcam ab150077) and/or anti-mouse Alexa Fluor 594 (1:500; Abcam ab150116). Nuclei were counterstained with DAPI (4,’6-diamidine-2-phenylindole dihydrochloride, Roche 10,236,276,001) dye and mounted with Prolong Gold anti-fade reagent (Invitrogen, P36930). Images were processed and analysed using confocal microscopy linked to the ZEN Imaging Software-LSM710. Post-confocal quantification and size measurement of the nuclear bodies were conducted using the ImageJ Software (NIH; https://imagej.nih.gov/ij/).