Irdye 800 goat anti rabbit igg

The protein levels of OAT1 and
BCRP were determined by Western blotting using 9% (W/V) sodium dodecyl
sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Cell samples
were homogenized in ice-cold Tris-Sucrose (TS) buffer (10 mM Tris-HEPES
and 250 mM sucrose, pH 7.4) containing protease inhibitors (PMSF 100
μM, Aprotinin 5 μg/mL, Leupeptin 5 μg/mL, Pepstatin
1 μg/mL, E64 10 μM). Membrane fractions were obtained
by high shear passage using a microfluidizer LV1 (Microfluidics, Westwood,
AM, USA), and cell lysates were span for 20 and 90 min at 4000 and
25 000 rcf at 4 °C, respectively. Membranes were incubated
with mouse anti-BCRP (1:200 dilution; Abcams, Cambridge, UK) and rabbit
anti-OAT1 antibody (1:100 dilution; Abcams, Cambridge, UK) overnight
at 4 °C. As a loading control, rabbit anti-Na, K-ATPase antibody
(α-subunit, 1:4000 dilution, C356-M09,^{27 (link)}) was used. Secondary antibodies, Alexa fluor 680 goat antirabbit
IgG (1:10 000 dilution; Life Technologies Europe BV), streptavidin
Alexa fluor 680 (1:10 000 dilution; Life Technologies Europe
BV), and IRDye 800 goat antirabbit IgG (1:10 000 dilution;
Rockland, PA). Fluorescence was detected using the Odyssey scanner
CLx (Li-Cor Biosciences, USA). Data were normalized to protein expression
levels of the loading control using ImageJ software (imagej.nih.gov).

Protein was extracted, quantified, and probed with antibodies, as described.^{40 (link)} Whole cell lysates were extracted using RIPA buffer (Cell Signaling). Antibodies used included p53 (Cell Signaling, Danvers, MA), Mdm2 (EMD Millipore, Billerica, MA), WIP1 (Bethyl, Montgomery, TX), and β-actin (Sigma-Aldrich, St. Louis, MO). Secondary antibodies Alexa Fluor 680 goat anti-mouse IgG (Life Technologies) or IRDye 800 goat anti-rabbit IgG (Rockland, Gilbertsville, PA) were used at a dilution of 1:5,000. Immunoblots were imaged as previously described.^{29 (link)}