Imager chemi doc xrs imaging system

For all other western blot analysis, proteins were separated on SDS polyacrylamide gels, and western blot analysis was carried out using standard procedures and the following antibodies: FlagM2 (F1804, Sigma; diluted 1:200), IQGAP1 (ab33542, Abcam, Cambridge, MA; diluted to 1 μg/ml), phospho-S6 kinase (Thr-389) (9234, Cell Signaling Technology; diluted to 1:1000), total S6 kinase (2708, Cell Signaling Technology; diluted to 1:1000), phospho-Akt (Thr-308) (4056S, Cell Signaling Technology; diluted to 1:1000), phospho-Akt (Ser-473) (4060S, Cell Signaling Technology; diluted to 1:1000), pan-Akt (4691, Cell Signaling Technology; diluted to 1:1000), peroxidase-conjugated anti-beta actin (A3854, Sigma; diluted 1:10,000), and horseradish peroxidase conjugated goat anti-mouse secondary (Jackson; diluted 1:2000). Visualization was performed using SuperSignal West Pico Chemilumnescent Substrate or SuperSignal Femto Chemiluminescent Substrate (Thermo Fisher Scientific), per the manufacturer’s instructions. Densitometry of bands was performed using a Bio Rad Molecular Imager Chemi Doc XRS+ Imaging System and ImageJ software.

The renal cortex was lysed with RIPA buffer (pH 7.5) (Pierce, IL, USA). Equal amounts (30 μg) of protein were separated on a 10% SDS-PAGE (100 V, 1.5 h), and transferred to a PVDF membrane (200 mA, 40 min) under ice-cold conditions. The membrane was blocked with 5% skim milk in Tris-buffered saline containing 0.1% Tween 20 for 1 h and incubated overnight at 4 °C with the primary antibodies TGF-β1 (1:500, ab92486, Abcam, UK) and MyD88 (1:500, CN89330, Bioworld, USA). After washed with Tris-buffered saline Tween 20 (TBST) and incubated for 2 h with horseradish peroxidase (HRD) -coupled goat anti-rabbit secondary antibody. The membrane was placed on molecular Imager chemi Doc™ XRS+ imaging system (Bio-Rad) for detection. The α-Tublin was used as an internal control. The density of the bands was measured using IPP.

CYP3A-like and CYP2-like protein levels were determined by Western blot as described in Maldonado et al. [25 (link)]. The membrane was probed with either 1:500 dilution (v/v) of primary rabbit anti-rainbow trout polyclonal CYP2K1 and/or CYP2M1 or 1:1000 dilution (v/v) of primary rabbit anti-rainbow trout polyclonal CYP3A27 antibodies provided by Dr. Malin Celander University of Gothenburg and Dr. Don Buhler, Oregon State University. Imager ChemiDoc XRS+ Imaging System (BioRad) image analyzer. The data were presented in optical density units per mg protein.