Pe conjugated goat anti mouse igg

Primary lung fibroblasts were cultured in a dish at 37 °C, and the mitochondria were stained with 100 nM MitoTracker Red (Invitrogen) for 20 min. Then, the cells were fixed with 4% (v/v) paraformaldehyde (PFA) for 45 min, permeabilized in ice-cold buffer (0.1% [w/v] sodium citrate and 0.1% [v/v] Triton X-100 in distilled water) for 3 min, blocked with Dulbecco’s phosphate-buffered saline with 1% (w/v) BSA for 1 h; and incubated with an anti-OAT antibody (1:200; Santa Cruz, #SC-374243) overnight at 4 °C. After washing, the cells were incubated with a fluorescein isothiocyanate (FITC)-conjugated anti‐mouse antibody (1:1000; Abcam, #ab6785) for 1 h at room temperature and mounted in fluorescence medium containing 4ʹ,6-diamidino-2-phenylindole (DAPI; Abcam). To measure the expression of TGF-β1 in BLM-treated OAT-knockdown cells, the cells were subjected to double immunofluorescence staining for TGF-β1 (1:100; Thermo Fisher Scientific, #MA5-15065) and OAT (1:100). FITC-conjugated donkey anti-rabbit IgG (1:1000; Abcam, #ab150061) and PE-conjugated goat anti-mouse IgG (1:1000; Abcam, #ab97024) served as the secondary antibodies. All the stained images were captured using a Leica DMi8 confocal laser microscope (Leica Microsystems; Wetzlar, Germany).

3 μL of the LABScreen® single antigen HLA bead suspension (One Lambda, Inc., CA, USA) was added per well of a 96-well plate and incubated with either 100 nM or 1 μM soluble WT TAPBPR, or with 1 μM TAPBPR TN5 mutant, at 22°C, with rotation, for 60 min. The beads were washed three times in wash buffer (One Lambda, Inc., CA, USA) to remove any excess of soluble TAPBPR and were then first incubated with PeTe4 antibody for 30 min, washed and then incubated with a PE-conjugated goat anti-mouse IgG (Abcam, UK) for another 30 min at 22°C. After a subsequent round of washing, cells were re-suspended in 1x PBS and the TAPBPR levels bound to the beads were measured by a using the Luminex Fluoroanalyser system (One Lambda, Inc., CA, USA) and analyzed using the HLA FusionTM software (One Lambda, Inc., CA, USA).