Odyssey clx fluorescent scanner

Western blot analysis was performed using samples of whole cell lysate or eluate from immunoprecipitation prepared in NuPage sample buffer supplemented with antioxidant reagent according to manufacturer’s specifications (Invitrogen). Samples were heated (10’ @ 70°C), then separated by electrophoresis in NuPage 4–20% polyacrylamide gels and transferred to nitrocellulose membanes using the NuPage XCell II Blow Module (Invitrogen). Blots were visualized using the Li-COR Odyssey CLX fluorescent scanner after blocking with fluorescent blocking buffer (Li-COR Biosciences), incubation with primary antibodies and fluorescently conjugated secondary antibodies (Li-COR Biosciences).
Primary antibodies: Mouse monoclonal–HNRNPC1/C2 (sc-32308, Santa Cruz), HNRNPU (3C6, Millipore), SRSF3 (7B4, Millipore), SRSF1 (AK96, Millipore); Rabbit polyclonal–FUSIP1 (A302-282A-1,Bethyl), ILF2 (A303-147A-1, Bethyl), IgG (2729, Cell Signaling), U2AF1 (A302-079A-1, Bethyl), SAM68/KHDRBS1 (sc-333, Santa Cruz), SFRS10 (AV40528, Sigma).

Yeast whole-cell protein samples were prepared using the TCA precipitation method described in O’Rourke and Herskowitz, 1998 (link). The proteins were resolved on the SDS-PAGE gel and transferred either onto PVDF membrane (Immobilon®-FL, 0.45-μm pores, Merck Millipore Ltd.) or nitrocellulose membrane (Protran BA83, 0.2 µm, Whatman). The proteins tagged with the C-terminal 4myc tag were detected using mouse monoclonal 9E10 antibody (Thermo Fisher Scientific 13-2500, 1:1000 dilution). Rfa1 was detected using rabbit polyclonal anti-RPA antibody (Agrisera AS07 214, 1:20,000 dilution). Act1 (the loading control) was detected using mouse monoclonal anti-beta Actin antibodies (Abcam ab8224, 1:50,000 dilution). GST was detected using mouse monoclonal anti-GST antibody (Abcam, 3G10/1B3). The secondary antibodies used for the western blotting were goat anti-mouse IgG (H + L) cross-adsorbed secondary antibody, Alexa Fluor 680 (Thermo Fisher Scientific A-21057, 1:12500 dilution) and goat anti-rabbit IgG F(c) IRDye800 Conjugated (Rockland 611-132-003, 1:12500 dilution), respectively. The membranes were scanned using Odyssey® CLx fluorescent scanner (LI-COR®) and analysed with the Image Studio™ Lite software.