Anti γh2ax ser139

Whole cell protein of untreated and 24 h Rapha Myr^® extract (0.5–1.25–2.5% v/v)-treated cells were prepared according to Laemmli (1970) and submitted to Western blot analysis, performed according to Grabowska et al., 2016 [72 (link)]. The primary antibodies used were: anti-integrin α5 (1:1000) (Immunological Sciences, Rome, Italy), anti-GAPDH (1:50,000) (Millipore, Darmstadt, Germany), anti-Poly (ADP-ribose)polymerase (PARP, 1:1000) (BD Biosciences, San Jose, CA, USA), anti-γH2AX Ser139 (1:1000) (Abcam, Cambridge, UK), anti-p53 (1:500) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-p21 (1:500) (Sigma-Aldrich, St. Louis, MO, USA), anti-phospho-p53 Ser15 (1:250), anti-sirt 1 (1:250), anti-phospho-sirt 1 Ser47 (1:250), anti-sirt 3 (1:500), anti-sirt 5 (1:500), anti-sirt 6 (1:1000) and anti-sirt 7 (1:250) (Cell Signalling Technology, Denvers, CO, USA). Each protein target was detected by using specific secondary horseradish peroxidase-conjugated antibodies (1:2000) (Dako, Glostrup, Denmark) and an ECL system (Thermo Scientific, Rockford, IL, USA). The expression level of proteins was measured by densitometric analysis using the software Image J and GAPDH was chosen as the reference protein.

Anti-phospho-AKT (Ser473), anti-AKT, anti-phospho-JAK2 (Tyr1007/Tyr1008), anti-JAK2, anti-phospho-Stat3 (Tyr705), anti-Stat3, anti-phospho-Chk2 (Thr68) and anti-phospho-p53 (Ser15) antibodies were purchased from Cell Signaling Technology (Boston, USA). Anti-Bax, anti-Bcl-2, anti-Mcl-1, anti-Caspase-3, anti-Caspase-9, anti-Survivin, anti-Cyclin A2, anti-phospho-ATM (Ser1981), anti-ATM, anti-γH2A.X (Ser139) and anti-LMP1 antibodies were purchased from Abcam (Cambridge, UK). Anti-pBad (S136) and anti-Bad were purchased from Bioworld Technology (Minneapolis, USA). Anti-p53 and anti-Zta antibodies were purchased from Santa Cruz Biotechnology (Texas, USA). Anti-β-actin and HRP-conjugated goat anti-mouse/rabbit secondary antibodies were purchased from ComWin Biotech (Beijing, China).