Phospho atm ser 1981

Cell lysates were prepared as described previously (Vitkeviciene et al., 2019 (link)). Proteins were fractionated in 7.5–15% SDS-PAGE gradient electrophoresis gel and transferred on the PVDF membrane. Primary antibodies against ATM (mouse, clone 6F-H2) (Thermo Fisher Scientific, Waltham, MA, United States), Phospho-ATM (Ser 1981) (Abcam) (dilution ratio 1:15000), SUZ12 (Cell Signaling Technology) (dilution ratio 1:1000), H3K27me3 (rabbit, polyclonal) (Millipore, Billerica, MA, United States), H3K14Ac (rabbit, polyclonal) (Millipore, Billerica, MA, United States), H4 hyper Ac (rabbit, polyclonal) (Millipore, Billerica, MA, United States), EZH2 (Cell Signaling Technology, Danvers, MA, United States) (dilution ratio 1:1000), GAPDH (mouse, clone 6C5) (Abcam, Cambridge, United Kingdom), HRP-conjugated secondary antibodies against mouse immuno-globulins (goat, polyclonal) (Agilent Dako, Santa Clara, CA, United States), and rabbit immunoglobulins (goat, polyclonal) (Agilent Dako, Santa Clara, CA, United States) were used according to the manufacturer’s instructions. GAPDH was used as a loading control. “Clarity Western ECL Substrate” (BioRad, Hercules, CA, United States) was used for chemiluminescent detection. Signal detection was carried out on ChemiDoc™ XRS+ System (BioRad, Hercules, CA, United States). Quantitative evaluation was performed using ImageJ software.

Antibodies for EMT, cell cycle, and DNA damage repair pathway were purchased as follows: ZEB-1 (CST:3396), N-CAD (CST:13116), E-CAD (CST:3195), Slug (CST:9585), Snail (CST:3879), Vimentin (CST:5741), β-catenin (CST:8480), CCND1 (CST:92G2), p21 (CST:2947 s), CDK4 (Abcam: Ab108357), CDK6 (Abcam:Ab124821), total ATM (CST:2873), phospho-ATM (Ser1981) (Abcam:5883), phospho-Chk2 (Thr68) (CST:2197), phospho-H2AX (Ser139) (CST:9718), RAD51 (Abcam:ab133534), and β-actin (CST:3700). All the western blot primary antibodies were 1:1000 diluted except RAD51 (1:10000 diluted). The antibody of HMGA2 (Proteintech, 20795-1-AP) was used in Immunohistochemistry (IHC).

Slices measuring 3–5 µm were cut from paraffin-embedded, formalin-fixed liver pieces. Slices were mounted on microscope slides (Superfrost Ultra Plus Adhesion Slides, Thermo Fisher Scientific). For RNAPII staining, samples were deparaffinized with xylene, rehydrated with an alcohol gradient and washed with Milli-Q water before antigen retrieval (30 min in citrate buffer, pH 6). The antibodies used were: Alexa Fluor 594-RPB1 antibody (in 1:250 dilution), recognizing all forms of RNAPII independently of the phosphorylation status of their CTD (cat. no. 664908, BioLegend); RNAPII-ser2p (cat. no. ab5095, Abcam); RNAPII-ser5p (cat. no. ab5131, Abcam); phospho-ATM (Ser1981, cat. no. 4526, Cell Signaling Technology); and phospho-histone H2A.X (Ser139, cat. no. 9718; Cell Signaling Technology) all in 1:500 dilution. To reduce the background fluorescence level, a mouse-on-mouse detection kit was used (cat. no. BMK-2202, Vector Laboratories). Sections were counterstained using DAPI or Hoechst 33342.