Mouse anti rfp 6g6

For ER chaperone secretion assays, HeLa S3 cells were transfected with 0.5 µg mScarlet-ligand (+xDEL ligand) or 0.5 µg pcDNA3.1 (− ligand), and allowed to express the respective proteins for 24 hr. The media were TCA precipitated and both cell and media were resuspended and boiled in SDS-PAGE sample buffer. All samples were analysed by Western blotting (Trans-Blot Turbo transfer system, Bio-Rad) for xDEL ligand (mouse anti-RFP 6G6, Chromotek), resident ER chaperones BIP (rabbit #ab21685, Abcam), PDI (rabbit #11245–1, ProteinTech), ERP72 (rabbit #5033S, Cell Signalling Technology), ERP44 (rabbit #3798S, Cell Signalling Technology) and the KDEL receptor (mouse ADI-VAA-PT048, Enzo Life Sciences). HRP-conjugated secondary antibodies and the ECL reagent were used to detect signals on film. Films were then digitised and signals measured by densitometry in FIJI (Schindelin et al., 2012 (link)). Data were plotted as bar graphs in GraphPad Prism.

Parasite protein samples of schizont stages (40–44 hpi) were prepared by saponin-lysis of infected erythrocytes and separated on a 12% SDS-PAGE gel at 120 V for 2 h. Separated proteins were transferred to a nitrocellulose membrane (LI-COR) for 1 h at 90 V and 4 °C using a tank blot device (Bio-Rad) according to the manufacturer’s instructions. Membranes were blocked with 5% milk in TBS followed by incubation with primary antibody diluted in 2.5% milk in TBS-T at 4 °C overnight. Primary antibodies were diluted as follows: mouse-anti-GFP (Roche) 1:2000; rabbit-anti-GFP (Thermo Scientific) 1:1000; mouse-anti-RFP (6G6, ChromoTek) 1:2000; and rabbit-anti-BiP (68 (link)) 1:2000 in 2.5% milk in TBS-T. After incubation with primary antibodies, membranes were 3× washed with TBS-T and incubated with secondary antibody for 1 h at room temperature in the dark. Secondary antibodies were diluted as follows: goat-anti-mouse800CW (LI-COR) 1:20,000; goat-anti-rabbit800CW (LI-COR) 1:20,000; goat-anti-mouse680RD (LI-COR) 1:20,000; and goat-anti-rabbit680RD (LI-COR) 1:20,000 in 2.5% milk in TBS-T. Membranes were washed again three times with TBS-T and analyzed in an Odyssey FC Imager (LI-COR).