Odissey fc imaging system

Cells were lysed in ice-cold whole cell extract buffer containing 50 mM TrisHCl at pH 7.4, 250 mM NaCl, 0.1% Nonidet NP40, 5 mM EDTA and NaF 50 mM with a protease inhibitor cocktail (Sigma). Lysates were cleared by centrifuging at 12,000 rpm for 5 min. Cell lysates containing equal amounts of protein (30–70 µg) were resolved on 10–12% SDS-PAGE (polyacrylamide gel electrophoresis) gels. The proteins were then transferred to nitrocellulose membranes (PROTRAN, Schleicher and Shull). Immunoblotting was carried out with the following antibodies and visualized using Odissey FC Imaging System (Li-COR): anti-PLK1 (F-8) #sc17783, anti-actin (C-11) #sc1615 and anti-beta tubulin #sc9104 were provided by Santa Cruz Biotechnology. Anti-phospho-Histone H3 (Ser10) (6G3) #9706 was purchased from Cell Signaling Technology and anti-H2AX pSer139 #05-636 was purchased by Millipore. The horseradish peroxidase (HRP) conjugated secondary antibody anti-goat (sc-2354) was purchased from Santa Cruz Biotechnology (Heidelberg, Germany). The anti-rabbit (#1706515) and anti-mouse (#1706516) antibodies were purchased from BIO-RAD LABORATORIES S.r.l.

Proteins extracted from the frozen specimens were homogenised in protein lysis buffer, loaded on SDS-PAGE and immunoblotted as previously described.^{10 (link)} An Odissey FC Imaging System (LI-COR) was used for acquisition. Primary anti-CCAAT Enhancer Binding Protein Alpha (C/EBPα)(1:150, cat.#sc-9314) and actin (1:500, cat.#sc-1616) were purchased from Santa Cruz Biotechnology (Heidelberg, Germany). Primary anti-UVSSA (1:500, cat.# A09174) was purchased from Biocompare- BosterBio (Pleasanton, California, USA).

Cell pellets were lysed with the Cell Lytic M reagent (Merck Life Science S.L.U., Madrid, Spain) containing a protease inhibitor cocktail (Roche cOmplete™, Merck Life Science S.L.U., Madrid, Spain). Proteins in the crude lysates were quantified using the Pierce^TM BCA protein quantification kit (ThermoFisher Scientific, Madrid, Spain) following the manufacturer’s instructions. A total of 20 μg of whole-cell lysates were separated by SDS-PAGE and transferred onto PVDF membranes. Blots were probed using primary antibodies anti-SMC2 (Merck Life Science S.L.U., Madrid, Spain) and β-Tubulin (Invitrogen, ThermoFisher Scientific, Madrid, Spain). Proteins were detected using HRP-conjugated secondary antibodies (Dako, Palex Medical SA, Barcelona, Spain) and developed after appropriate incubation in an Immobilion^® Western reagent (Merck Life Science S.L.U., Madrid, Spain) using an Odissey FC imaging system (LI-COR Biotechnology GmbH, Bad Homburg, Germany) for chemiluminescence detection.