Irdye 800cw conjugated goat polyclonal anti mouse secondary antibody

NRCF were seeded either in 35 mm glass-bottom dishes as described above or in clear-bottom black-walled 96-well plates (655090; Greiner Bio-One, Monroe, NC) at 3 × 10⁴ cells/well and infected with Ad-Flag-EGFR-mYFP (constructed at Vector Biolabs, Philadelphia, PA) at an MOI of 200 for 24 h prior to stimulation with agonist as indicated. After 1 h, cells were fixed in 4% paraformaldehyde (163201145; Wako Chemicals, Richmond, VA) for 20 min, rinsed with phosphate-buffered saline (Corning/Cellgro, 21-030-CV) and on-cell western staining (nonpermeabolized) was performed using anti-Flag M2 antibody (1:1000, 3 h at RT, Sigma, F1804), IRDye® 800CW Conjugated Goat (polyclonal) Anti-mouse secondary antibody (1:1000, 1 h at RT, LI-COR, 926-32210) and DRAQ5 (1:5000, 1 h at RT, Cell Signaling Technology, #4084) following the LI-COR on-cell protocol. Flag and DRAQ5 signals were detected using Odyssey CLx infrared imaging system (LI-COR Biosciences, Lincoln, NE) and YFP signal was measured with a Tecan M1000 plate reader. Amount of EGFR internalization was calculated by normalizing the Flag signal to both the DRAQ5 (cell number control) and YFP (infection control) signals: Flag/DRAQ5/YFP.