Alexa 488 or 546 conjugated secondary antibodies

The CD8A‐tagged constructs internalization and recycling were performed as previously described.¹⁷ Briefly, Control or SNX27 KO HeLa cells were transfected with plasmids encoding CD8A‐tagged constructs. After 24 h of transfection. 5 µg/ml monoclonal antihuman CD8A antibody (Invitrogen, 14‐0086‐80) was added on ice for 30 min. Redundant antibodies were washed away by with ice‐cold PBS three times. The internalization of antibody‐bound CD8A‐tagged constructs complexes chase in DMEM at 37˚C for 30 min (detection the colocalization of CD8A with EEA1) or 60 min (detection the colocalization of CD8A with LAMP1). At indicated time points, cells were taken out for immunofluorescence staining with Alexa‐488 or 546‐conjugated secondary antibodies (Jackson), and then detected by confocal microscopy.

Internalization and recycling of CD8A-tagged constructs were performed as previously described [20 (link), 21 (link)]. Briefly, cytoplasmic tails of transmembrane proteins were fused to the C terminus of CD8A. HeLa cells were transfected with plasmids encoding CD8A-cargo fusion proteins for 24 h before the internalization assay. Monoclonal anti-human CD8A antibody (5 μg/mL in DMEM) was added on ice for 30 min. Unbound antibodies were removed by washing with ice-cold wash buffer (0.1 M glycine and 0.15 M NaCl [pH 3.0]) twice and PBS once. The internalization of antibody-bound CD8A-cargo complexes was carried out in DMEM at 37°C for 1 or 3 h. At indicated time points, cells were fixed with 4% PFA and permeabilized with 0.1% Triton X-100. The internalized CD8A–antibody was detected using Alexa-488 or 546-conjugated secondary antibodies (Jackson).
To assess internalization and recycling of endogenous TRAILR1, HeLa cells were incubated with DMEM containing antibody against the extracellular domain of TRAILR1 (5 μg/ml) and lysosomal protease inhibitor leupeptin (100 μM). After incubation at 37°C for 6 h, unbound antibodies were washed away. Cells were then fixed and permeabilized, and the TRAILR1 antibody was detected by an Alexa-488 coupled secondary antibody (Jackson).