Rabbit anti calnexin

At the indicated time points, infected HeLa cells were washed three times with KHM buffer (110 mM potassium acetate, 20 mM HEPES, 2 mM MgCl₂, pH 7.3), and the plasma membrane selectively permeabilized by incubation with digitonin (40 μg/mL in KHM buffer) for 1 min at RT, followed by three washes with KHM buffer. Cells were then incubated for 12 min at RT with rabbit anti-Calnexin (Stressgen, 1:250 in KHM), to label the cytosolic face of the endoplasmic reticulum in permeabilized cells, and goat anti-Salmonella CSA1 antibodies (KPL, 1:100 in KHM), to detect cytosolic bacteria. Cells were washed in PBS, fixed in PFA and all host cell membranes were permeabilized with SS-PBS. Antibodies delivered post-digitonin permeabilization were detected with Alexa Fluor 647-conjugated anti-rabbit and Alexa Fluor 568-conjugated anti-goat antibodies. After washes with PBS, SopB^3xFLAG and SipA effectors were detected using mouse anti-FLAG M2 (Sigma-Aldrich, 1:250) and mouse anti-SipA (1:50), respectively. Cells were washed again in PBS and incubated with Alexa Fluor 488-conjugated anti-mouse to detect effector bound antibodies and Pacific Blue-conjugated goat anti-Salmonella CSA1 to label all intracellular bacteria. Coverslips were then washed sequentially with PBS and distilled water, and mounted on glass slides in a Mowiol solution.

CHO cells were seeded onto glass bottom, 96 well plates and transfected with RUSH-APOL1 plasmids. 24 hr after transfection, cells were treated with or without 80 µM biotin every 30 min for 0–120 min. For cell surface immunostaining, cells were moved onto ice and blocked with HBSS + Ca²⁺ + Mg²⁺ + 0.5% BSA fraction V, stained with primary antibodies, fixed in 2% formaldehyde (Thermo 28906), quenched with 50 mM NH₄Cl, and then stained with secondary antibodies. For intracellular staining, cells were permeabilized with 0.075% saponin. Staining was performed with the following antibodies and dyes: mouse anti-APOL1 1:800, rabbit anti-calnexin 1:200 (Stressgen, Farmingdale, NY. SPA-860), anti-rabbit Alexa 488 plus 1:1500 (Thermo A32731), anti-mouse Alexa 647 1:1000 (Thermo A21236), and HCS Nuclear Mask 1:400 (Thermo H10325). Cells were imaged via spinning disk confocal microscopy.