Pvdf fl

Half of the quadriceps muscle was solubilized in 1 ml of tris-buffered saline (TBS) containing 1% Triton X-100 and protease inhibitors. The solubilized fraction was incubated with 200 μl of WGA–agarose bead slurry (Vector Laboratories, Burlingame, CA) overnight at 4°C. Pellets formed from the beads and were washed three times in 1 ml of TBS containing 0.1% Triton X-100 (15 (link)). The beads were then either directly mixed with SDS–polyacrylamide gel electrophoresis (PAGE) loading buffer (for Western blotting, ligand overlay) or eluted with 1 ml of TBS containing 0.1% Triton X-100 and 300 mM N-acetylglucosamine (for solid-phase binding assay). Proteins were separated by 3 to 15% SDS-PAGE and transferred to polyvinylidene difluoride–FL (PVDF-FL; Millipore Sigma) membranes. The membranes were incubated with a sheep polyclonal antibody to human DG (1:100 dilution; R&D Systems, AF6868) and a mouse monoclonal antibody to a glycoepitope on the sugar chain of α-DG (IIH6; 1:100 dilution) followed by IRDye 800CW dye-conjugated goat anti-sheep IgG (LI-COR, 926-32214) and goat anti-mouse IgM (LI-COR, 926-32280), respectively.

Nucleus and cytoplasm-enriched protein lysate fractions were isolated from homogenized mouse ovaries and protein concentration was quantified using the Biorad DC Protein Assay (Bio-Rad, Hercules, CA) according to manufacturer’s instructions. Protein fractions (10–20 μg per lane) were prepared in Laemmli Sample Buffer, heated 10 min at 65°C, size-separated by SDS-PAGE, transferred to PVDF-Fl (Millipore, Billerica, MA) membranes, and pre-blocked in TBS-T (0.05% Tween-20) + 5% BSA for 1 h at room temperature as previously described [23 (link)]. Membranes were incubated overnight at 4°C in TBS-T + 5% BSA containing polyclonal rabbit anti-S139-phosphorylated γH2AFX (1:500, catalogue number ab11174, Abcam, Cambridge, MA) and monoclonal mouse anti-β actin (1:10,000, catalogue number A5316, Sigma, St. Louis, MO) as a loading control. Blots were washed with TBS-T then incubated simultaneously for 1 h at room temperature with donkey anti-rabbit Alexa 680 (1:15,000 in TBS-T; catalogue number A10043, Molecular Probes, Grand Island, NY) and donkey anti-mouse IRDye 800 (1:15,000 in TBS-T; catalogue number 926–32212, LiCor, Lincoln, Nebraska). Blots were washed with TBS-T, dried, scanned and quantified using the LiCor Odyssey System (University of Wisconsin-Small Molecule Screening Facility) and Odyssey software.