Anti goat sc 2354

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.

HEL cells and leucocytes from MPN patients were used for immunoblotting assays. Total proteins were isolated with radioimmunoprecipitation assay (RIPA) buffer and quantified as previously described.²⁸ 70 µg of each cell lysate was loaded, resolved through SDS‐PAGE 6%‐12% gel and electroblotted onto 0.2 µm PVDF membranes (GE Healthcare, Chicago, IL, USA). After blocking with 5% non‐fat milk (Sigma‐Aldrich, Thermo Fisher Scientific, Waltham, MA, USA), membranes were incubated 2 hrs at RT with Bcl‐xL (H‐5 sc‐8392) or overnight at 4°C with Phospho‐Jak2 (Tyr) (3771, Cell Signaling Technology, Danvers, MA, USA), JAK2 (C‐14 sc‐34479), GAPDH (A‐3 sc‐137179) and β‐Actin (C4 sc‐47778) [Santa Cruz Biotechnology, Dallas, TX, USA] primary antibodies with a dilution of 1:2000. Then, membranes were incubated with specific horseradish peroxidase (HRP)‐conjugated secondary antibodies: anti‐rabbit (sc‐2357), antimouse (sc‐2005) and anti‐goat (sc‐2354) [Santa Cruz Biotechnology, Dallas, TX, USA] in a dilution of 1:7000 for 1 hour at RT. Immunoreactive bands were visualized by Clarity Western ECL Substrate (Bio‐Rad, Hercules, CA, USA) and acquired by the Image Lab program (Bio‐Rad, Hercules, CA, USA).

Cell pellets were lysed for 30 min in ice-cold whole cell extract buffer (50 mM TrisHCl pH 7.4, 250 mM NaCl, 0.1% Nonidet NP40, 5 mM EDTA, 50 mM NaF and a protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA). Lysates were cleared by centrifuging at 12,000 rpm for 5 min and the protein concentration was determined using a BioRad assay kit (BioRad, Hercules, CA, USA). Cell lysates (50µg) were resolved on 10–12% SDS-PAGE (polyacrylamide gel electrophoresis) gels. Proteins were then transferred to nitrocellulose membranes (Merck Millipore, Burlington, MA, USA). Immunoblotting was carried out with the following antibodies: rabbit anti-PARP #9542 (cell signaling, 1:1000), rabbit anti-γH2AX #9718 (Cell Signaling, Danver, MA, USA, 1:1000), goat anti-ACTIN sc-1615 (Santa Cruz Biotechnology, Dallas, TX, USA, 1:500) and mouse anti-RAN sc-271376 (Santa Cruz Biotechnology, 1:500). The secondary antibodies conjugated with horseradish peroxidase (HRP) anti-rabbit #1706515 and anti-mouse #1706516 were purchased from BIO-RAD Laboratories S.r.l., anti-goat sc-2354 from Santa Cruz Biotechnology. HRP substrate (ECL Western Blotting Detection, Amersham-Life Science, Amersham, UK) was added and the signal was detected with the Odyssey Fc instrument (Li-COR). All the uncut filter and relative densitometric raw data have been included in Figure S7.