Veriblot reagent

For immunoprecipitation experiments, cells were lysed in 140 mM NaCl, 10 mM Tris pH 7.6, 1% Triton X-100, 0.1% sodium deoxycholate, 1 mM EDTA, protease and phosphatase inhibitors. Cells were incubated on ice for 30′, then centrifuged for 20′ at 4 °C, at 13000 rpm. Thereafter, supernatant (1–2 mg) underwent immunoprecipitation as described^{33 (link)}, by using 4 μg of each antibody or control IgGs (mouse IgGs were from Santa Cruz, cat. #sc-2025; rabbit IgGs were from Merck, #cat. I8140). To avoid IgGs detection, either ImmunoCruz IP/WB Optima B, C, F, E (Santa Cruz Biotechnology; cat. #sc-45039, cat. #sc-45040, cat. #sc-45043, cat. #sc-45042, respectively) or the VeriBlot reagent (Abcam; #cat. ab131366 and #ab131368) were used in western blot, after the immunoprecipitation procedure, according to the manufacturer’s instructions.

Cell pellets were resuspended in lysis buffer (20 mM Tris–HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100 (Sigma-Aldrich), protease/phosphatase inhibitors cocktail (Roche) and 20 nM N-ethylmaleimide (NEM) (Sigma-Aldrich) to prevent deSUMOylation [38 (link)], sonicated and incubated for 15 min on ice. BCA protein assay (Thermo Fisher Scientific) was used to quantify protein lysates and 30 μg protein samples were run on 10% NuPAGE Bis–Tris pre-cast polyacrylamide gels (Thermo Fisher Scientific) by SDS-PAGE and transferred to nitrocellulose membranes. Immunoblots were performed with specific primary antibodies (listed in Supplementary Table 1), diluted in 5% milk in TBS with 0.1% Tween-20 (Sigma-Aldrich). The HRP-conjugated secondary antibodies were detected by the Clarity ECL kit (Biorad), while the Veriblot reagent (Abcam, Cambridge, UK) was used to avoid interference of denatured IgG chains in immunoprecipation detection assays (see below). Densitometric analyses were performed using ImageJ software (NIH).