Goat anti rabbit irdye800rd

Protein was extracted from 5 ml of log-phase culture using a TCA/NaOH precipitation as described in Volland et al. [57 (link)]. Equal concentrations of protein (25 μg) were run on a NuPAGE 4–12% Bis-Tris gel (Invitrogen, #NP0323BOX) and transferred to a nitrocellulose membrane (Bio-rad 0.2 µm, #LC2009). Primary antibodies used for western blotting: rat anti‐FLAG (Agilent, #200474), mouse anti‐PGK1 (Abcam, #Ab113687), mouse anti‐Rpb3 (Biolegend, #665004), rat anti-S2P (Chromotek, #3E10), rat anti-S5P (Chromotek, #3E8), Rabbit anti-Spt5-P (#1761, kind gift from Prof. Steven Hahn) [27 (link)] and Rabbit anti-Spt5 (kind gift from Prof. Grant Hartzog) [58 (link)]. Secondary antibodies used for western blotting: goat anti-mouse IRDye680RD (LI-COR, #926-68070), goat anti-rat IRDye800RD (LI-COR, #926-32219), goat anti-rabbit IRDye800RD (LI-COR, #925-32211). The Odyssey infrared imaging system (LI-COR Bioscience) was used to visualize proteins-of-interest, and signals quantified by the median method of the Odyssey software. For quantitation, data were normalized against the PGK1 signal unless otherwise stated in the figure legend.