Alexa fluor 594 f ab 2 fragment of goat anti mouse igg

HUVECs were grown in 4-well chambered slides overnight and treated with GT3 or vehicle (DMSO) for 1 h or 24 h before irradiation. Cells were washed with PBS and fixed with 4% paraformaldehyde 1 h after irradiation. The fixed cells were washed, permeabilized with 0.3% Triton-X for 15 min at room temperature, quenched with 1% glycine, and blocked with 4% BSA in 1X TBST overnight at 4°C. The cells were incubated with primary antibody (1:600 dilution; monoclonal Anti-phospho-Histone H2AX (Ser139), clone JBW301; Millipore, Temecula, CA) for 3 h at room temperature. After washing 3 times with PBS, secondary antibody (Alexa Fluor 594 F (ab') 2 fragment of goat anti-mouse IgG; Invitrogen, Grand Island, NY) was added, and the cells were incubated for 1 h at room temperature in the dark. Finally, the cells were washed with PBS and stained with Hoechst solution to visualize nuclei. γ-H2AX foci were detected with a Zeiss AxioPlan microscope. γ-H2AX foci were not counted in apoptotic cells. The apoptotic cells, particularly cells in late apoptotic stages, were identified by their highly compromised nuclear membrane.

Antibodies against the following proteins were used in this study: GAPDH (Santa Cruz, Dallas, TX, USA), LAMIN B1 (MBL, Tokyo, Japan), PARIS (Millipore), PARIS (Proteintech, Chicago, IL, USA), PGC-1α (Millipore), PDGFRα (R&D, Minneapolis, MN, USA), PPARγ (Santa Cruz) HRP-conjugated F(ab’)2 fragment of goat anti-rabbit IgG (Jackson ImmunoResearch), HRP-conjugated F(ab’)2 fragment of goat anti-mouse IgG (Jackson ImmunoResearch), HRP-conjugated F(ab’)2 fragment of donkey anti-goat IgG (Jackson ImmunoResearch), Alexa Fluor® 594 F(ab’)2 fragment of goat anti-mouse IgG (Invitrogen, Waltham, MA, USA), Alexa Fluor™ 488 F(ab’)2 fragment of goat anti-mouse IgG (H + L) (Invitrogen), and Alexa Fluor™ 594 F(ab’)2 fragment of goat anti-rabbit IgG (H + L) (Invitrogen).