Fig. 1.

Miniaturized western blot setup to perform a screening of PCNA ubiquitylation inhibitors. a U2OS cells were UV irradiated (15 J/m²) and treated for 12 h with the proteasome inhibitors Epoxomicin and MG-132. The western blot was performed with two monoclonal antibodies to simultaneously detect total PCNA (in red) and ubi-PCNA (in green) using a LI-COR Odyssey infrared scanner. The ratios of ubi-PCNA/total PCNA were normalized to the highest induction of ubi-PCNA in the non-treated (NT) UV-irradiated sample. b Three days detailed protocol to screen for PCNA ubiquitylation inhibitors, showing the quality controls to ensure reproducibility and robustness of PCNA ubiquitylation induction: (i) use of an infrared scanner to confirm the homogenous distribution of cells in the wells across the entire plate before the addition of the screening compounds; (ii) Automatized capture of a low magnification brightfield image at the center of each well as a control of the general cytotoxicity of every treatment; (iii) Lysis in benzonase w/o boiling of the samples and direct loading of the samples to the SDS Page gel. c Layout of the 96 multi-well (MW) plates used in the screening, showing the disposition of the non-irradiated and UV-irradiated controls. Eighty kinase inhibitors per plate were evaluated and eight mini-western blots were run in parallel with the 12 samples from each plate row. d Results of the screening with 627 kinase inhibitors from the PKIS2 library, tested at 1 μM. The distribution of the normalized ubi-PCNA/total PCNA ratios is shown. The dotted line represents the threshold of three standard deviations that allowed the identification of 22 hits