Rabbit anti ha polyclonal antibody

An HA-tag enzyme-linked immunosorbent assay (ELISA) was performed to confirm expression of wildtype and mutant PGT variants stably transfected into HEK-JumpIn cells upon induction with 1 μg/mL doxycycline. HEK-JumpIn-SLCO2A1 wildtype, A396E, A396T or L563P were seeded (60.000 cell/well) on a poly-D-lysine coated, transparent, flat-bottom 96 well plate in medium with or without 1 μg/mL dox and grown for an addition 30 h at 37°C 5% CO₂. Cells were washed with PBS, fixated for 10 min with 4% formaldehyde and permeabilized and blocked using Tris-buffered saline (TBS) with 2% BSA and 0.2% saponin for 1 h. Next, cells were incubated with 1:2500 primary Rabbit anti-HA polyclonal antibody (Invitrogen, Carlsbad, CA, United States) in assay buffer (TBS with 0.1% BSA) for 1 h at RT. Cells were washed with assay buffer prior to incubation with 1:6000 Goat-anti-rabbit HRP-conjugate antibody (Brunschwig Chemie, Amsterdam, Netherlands) in assay buffer for 30 min at RT. Immunoreactivity was detected by incubating with 3,3′,5,5′-tetramethylbenzidine (TMB) for 2 min before quenching with equal volume of 1M H₃PO₄. Absorbance was measure at 450 nm using a Wallac EnVision multimode plate reader (PerkinElmer, Groningen, the Netherlands).

JumpIn-DAT cells were grown in culture medium to 80% confluence. Cells were trypsinized, counted and seeded in a sterile 96-well flat bottom plate in culture medium at 60,000 cells/well in the presence of increasing amounts (1 pg/ml–1 µg/ml) of dox (100 µl total volume). Cells were incubated at 37 °C and 7% CO₂ for 24 h. All subsequent handlings were performed at room temperature. After incubation, cells were washed with PBS and fixed with 3.7% formaldehyde for 10 min. Cells were washed with DMEM and blocked with DMEM containing 2% (w/v) BSA and 0.2% (w/v) saponin for 1 h. After blocking, cells were incubated with 1:2500 rabbit anti-HA polyclonal antibody (Invitrogen, Carlsbad, CA, USA) for 30 min. Subsequently, cells were washed with DMEM containing 25 mM HEPES and incubated with 1:3000 goat anti-rabbit HRP-conjugated IgG antibody (Brunschwig Chemie, Amsterdam, The Netherlands) for 30 min. Immunoreactivity was visualized and measured as described in the “Whole-cell FLAG-tag ELISA” section.