Irdye 680rd goat anti mouse dyes

Cells were harvested as we have described [13 (link), 33 (link), 34 (link)] except for experiments to measure sumoylation where iodoacetemide (10 mM) was added to the lysis and wash buffers to preserve SUMOylation. Western blots were performed as described [33 (link), 35 , 36 (link)]. Briefly, 50 μg of protein was resolved by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with primary antibody (1/1000 dilution) and secondary antibody (1/10000 dilution). Bound antibody was detected with the LI-COR system. Blots were incubated with IRDye® 680RD Goat Anti-Mouse Li-COR dyes and visualized with an Odyssey® CLx Imaging System (LI-COR, Inc., Lincoln, NE) using LI-COR Odyssey software. Band intensities were quantified using the Quantity One software (Bio-Rad, Hercules CA) and intensities normalized to loading control as previously described [34 (link)].

The following antibodies were used. Anti-SERT rabbit polyclonal antibody/AB10514P (EMD Millipore, Billerica, MA); anti-P-gp mouse monoclonal antibody Clone 2F7 (OriGene, Rockville, MD); anti-MRP3 rabbit anti-human antibody LS-C177398 (Lifespan Biosciences Inc, Seattle, WA); anti-NET rabbit polyclonal antibody LS-C101935 (Lifespan Biosciences Inc, Seattle, WA) and anti-BCRP mouse monoclonal antibody BXP-21 (Abcam, Cambridge, MA). Loading control antibodies included: Anti-GAPDH (6C5, sc-32233, Santa Cruz Biotechnologies, Santa Cruz, CA); mouse monoclonal Grb2 (BD Biosciences, San Jose, CA), and anti-α-Tubulin clone B512 (Sigma-Aldrich Co, St. Louis, MO). Primary antibodies were diluted according to the manufacture’s suggestion (1:1,000). IRDye® 800CW Goat Anti-Rabbit and IRDye® 680RD Goat Anti-Mouse Li-COR dyes (LI-COR, Inc., Lincoln, NE) were diluted at 1:20,000. RFU readings were normalized to control housekeeping proteins (Grb2, Tubulin, or GAPDH) in qWestern-blot assays.