Mouse monoclonal anti nf ya antibody

E. invadens trophozoites and cysts were fixed with acetone/methanol (1:1) and permeabilized with 0.1% Triton X-100. Cells were incubated with 3% bovine serum albumin (BSA) for blocking followed by mouse monoclonal anti-NF-YA antibody (Santa Cruz; catalog no. sc-17753) (1:200) or anti-NF-YC antibody (Abcam; catalog no. ab232909) from rabbit (1:500) followed by Alexa Fluor 488 anti-mouse or anti-rabbit antibody, respectively (Molecular Probes) (1:2,500). Localization was performed with anti-DDX4 antibody (Thermo Fisher) from mouse (1:200), followed by Alexa Fluor 543-conjugated anti-mouse secondary antibody (Molecular Probes) (1:2,500). Slides were prepared using Vectashield mounting medium with DAPI (4′,6-diamidino-2-phenylindole; Vector Laboratories, Inc.) and visualized using a Leica CTR6000 microscope and a BD CARVII confocal unit. Images were analyzed using Leica LAS-AF software.

E. invadens trophozoites or cysts at particular time points (24 h, 48 h, and 72 h) were collected and lysed in lysis buffer. Briefly, the cells were lysed by sonication (1 pulse of 10 s for Trophs and 5 pulses of 10 s each for cysts) in lysis buffer containing protease inhibitors (PIC) (1× PIC, 1 mM leupeptin, 1 mM E-64). Protein lysate was resolved by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) for immunoblotting. The membranes were blotted with antibodies against mouse monoclonal anti-NF-YA antibody (Santa Cruz; catalog no. sc-17753) (1:1,000), rabbit polyclonal anti-NF-YC antibody (Abcam; catalog no. ab232909) (1:1,000), mouse monoclonal anti-DDX4 antibody (Thermo Fisher; catalog no. 2F9H5) (1:2,000), and rabbit polyclonal anti-beta-actin antibody (Abcam; catalog no. ab227387) (1:10,000). Horseradish peroxidase (HRP)-conjugated secondary antibodies against mouse or rabbit were used at 1:10,000 dilution for 1 h at room temperature and signal detected with ECL⁺ (GE).