Amersham protran premium 0.45 nc nitrocellulose blotting membrane

Samples were separated on Novex 4–12% gradient gels (Thermo Fisher Scientific) using NuPAGE® MOPS SDS running buffer (50 mM MOPS, 50 mM Tris-base, 0.1% SDS, 1 mM EDTA pH 7.7) or in-house casted gels of different Acrylamide percentages and transferred onto Amersham Protran Premium 0.45 NC Nitrocellulose blotting membranes (GE Healthcare) using a Bolt Mini-Gel system (Thermo Fisher Scientific), which was used for both the gel electrophoresis and the protein transfer to the membrane according to vendor instructions. Membranes were stained with Ponceau-S (Sigma-Aldrich) to determine total amount of protein loaded. Next, membranes were blocked with blocking solution (8% milk, 0.1% Tween-20 in PBS) for 1 h prior to primary antibody incubation. Primary antibodies were incubated overnight and secondary antibodies for 1–2 h, except for the PCNA-Ub immunostaining, where overnight incubation with the secondary antibody was also performed. Chemiluminescence reaction was initiated with Western Bright Quantum Western blotting detection kit (Advansta-Isogen) and measured in a ChemiDocTM imaging system (BIO-RAD, Hercules, CA, USA), or an ImageQuant 800 (Cytiva, Malborough, MA, USA). Antibodies are listed in Supplementary Table 2.

Cells were lysed in 2% SDS, 1% NP-40, 50 mM Tris pH 7.5, and 150 mM NaCl, unless stated otherwise. Proteins were separated on 4-12% gradient gels (Bold, Thermo Fisher Scientific) using MOPS buffer or 3-8% gradient gels (NuPage, Thermo Fisher Scientific) using Tris-Acetate buffer. Proteins were subsequently transferred onto Amersham Protran Premium 0.45 NC Nitrocellulose blotting membrane (GE Healthcare), using a submarine system. Membranes were stained with Ponceau-S to visualize total protein and blocked with PBS containing 0.05% Tween-20 and 8% milk powder. Subsequently, membranes were stained with primary and secondary antibodies diluted in PBS containing 0.05% Tween-20 and 8% milk powder. ImageJ (v1.53f51) was used for signal quantification of protein bands, where applicable.