Secondary rabbit igg goat igg antibodies conjugated to horseradish peroxidase

Human ASCs were transfected with siRNA as described above and plated in 6-well plates at a density of 500.000 cells. At day 12 of differentiation, cells were lysed in RIPA buffer as described [24 (link)]. Total liver protein was obtained from Novus Biologicals (CO, USA). 20–25 μg of total protein was separated by SDS-PAGE and Western blot was performed according to standard procedures. The membranes were blocked in 3 % ECL Advance™ Blocking Agent (GE Healthcare, Buckinghamshire, UK). Primary antibodies were OCT1-rabbit IgG (ab181022, Abcam, Cambridge, UK) used at a concentration of 1:2500, OCT1-rabbit IgG (LS-C354446, LifeSpan Biosciences, Inc., WA, USA) (1:500), OCT3-goat IgG (42–915, ProSci Inc., CA, USA) (1:500), MATE1-rabbit IgG (#14550, Cell Signaling Technologies, Danvers, MA, USA) (1:1000), PMAT-rabbit IgG (HPA052829, Atlas AB, Bromma, Sweden) (1:200); GAPDH-rabbit IgG (1:2000) was used as a loading reference (Cell Signaling). Secondary rabbit-IgG/goat-IgG antibodies conjugated to horseradish peroxidase were used (Sigma-Aldrich). Proteins were detected by chemiluminescence using ECL™ Select Western Blotting Detection Kit (GE Healthcare) in Chemidoc XRS system (Bio-Rad, Hercules, CA) and quantified by Quantity One software (Bio-Rad).