Fitc conjugated anti flag antibody

Transfectants were blocked by incubating 1 × 10⁶ cells for 15 min on ice in 100 μL 1% PBS-A (albumin in phosphate-buffered saline). Surface antigens were stained by incubating for 1 h on ice with 1 μg/ml FITC-conjugated anti-FLAG antibody (Sigma-Aldrich) and 1 μg/mL phycoerythrin-conjugated anti-rat CD2 (AbD Serotec, Kidlington, Oxfordshire, UK) in 100 μL PBS-A. Cells were washed three times in PBS and fixed with 4% paraformaldehyde in PBS for 15 min at room temperature. Cells were washed three times in PBS and permeabilized in 0.1% saponin (Sigma-Aldrich) in PBS-A. Intracellular antigens were stained by incubating for 1 h at room temperature with 1 μg/mL Alexa Fluor 647-conjugated anti-mouse Lck (BD Biosciences, San Jose, CA) and 1 μg/mL Pacific Blue-conjugated anti-mouse ZAP-70 (Thermo Fisher Scientific, Guilford, CT) in 100 μL PBS-A. Cells were washed three times with PBS and analyzed using a FACScan flow cytometer (Becton Dickinson, Franklin Lakes, NJ).

Cells were grown on 35 mm poly-D lysine-coated glass bottom dishes (MatTek, Ashland MA). For live cell microscopy, cells were transiently transfected with an expression plasmid for ERoxBFP (Costantini et al., 2015 (link)) (Addgene #68126, a gift from Erik Snapp (Costantini et al., 2015 (link))) and visualized at 24 – 48 hr post-transfection. For immunostaining, cells were washed with PBS, fixed with 2% paraformaldehyde, permeabilized with 0.1% Triton X-100 in PBS, blocked with 1% BSA and 0.1% Tween-20 in PBS, stained with FITC-conjugated anti-FLAG antibody (Sigma, St. Louis MO, F4049) and unconjugated rabbit antibodies against either calnexin (Cell Signaling Technology, Danvers MA, #2679), LMAN1 (Abcam, ab125006), or GM130 (Abcam, ab52649), then stained with Alexa647-conjugated anti-rabbit secondary antibody (Abcam, ab150075). All fluorescent imaging was performed on a Nikon A2 confocal microscope. Colocalization quantification was performed with Nikon Elements software. For all microscopy experiments, the observer was blinded to cell genotype and only unblinded after completion of quantitative analysis.