B7389

The preparation of whole cell lysates and western blot analysis of protein expression were performed according to the previous routine procedure [11 (link)]. In brief, total protein was extracted from H9C2 cells and protein concentration was determined using the Bicinchoninic Acid protein detection kit (Sigma-Aldrich). The whole cell lysis buffer was mixed with SDS loading buffer. After centrifugation at 13,000 g at 4 °C for 20 min, the supernatants were collected. Equal amounts of proteins (50 μg) were subjected to SDS-PAGE on 10 or 5% pre-casted gels (Bio-Rad, Hercules, CA, USA). The supernatant was collected after centrifugation at 13,000 g at 4 °C for 20 min. According to the previously established protocol, protein expression was detected [11 (link)]. The primary antibodies used were as follows: anti-SORBS2 (1:200, SAB4200183, Sigma-Aldrich), anti-ENH (1:200, SAB2101761, Sigma-Aldrich) and anti-actin (1:500, A5385, Sigma-Aldrich). The secondary antibody used were anti-mouse IgG (1:10000, A5385, Sigma-Aldrich), anti-rabbit IgG (1:10000, B7389, Sigma-Aldrich). Quantification was performed by measurement of the intensity of the signals with aid of ImageJ (NIH, Bethesda, MD, USA).

After deep anesthesia with ketamine/xylazine (90/10 mg/kg), animals were sacrificed by intracardiac perfusion with 4% paraformaldehyde in phosphate buffer as previously described [29 (link)]. Frozen hearts were cut into 14-micron sections and mounted on slides. Subsequently, the sections were dehydrated to perform the inhibition of endogenous peroxidase with H₂O₂ (0.5% v/v in methanol) for 30 min at room temperature; then rehydrated and permeabilized with 1% Triton X-100 in buffer phosphate saline (PBS) for 30 min. Nonspecific sites were blocked with 3% equine normal serum in PBS for 30 min, and incubation with anti-P-gp (dilution: 1/500; EPR10364-57 Abcam Inc., Cambridge, MA, USA) was performed for 48 h at 4 °C. Subsequently, incubation with a biotinylated secondary antibody (dilution 1/800; B7389 Sigma, St. Louis, MO, USA) was performed for 3 h at room temperature. Immunoreactivity was revealed with diaminobenzidine/nickel until coloration was observed and mounted with DPX (Distrene-Plasticizer- Xylene) mounting medium. Slides were also treated with monoclonal antibody anti-HIF-1α (dilution: 1/500 NB-100-131 NovusBio, Littleton, CO, USA) and revealed by immunofluorescence using a labelled Alexa488 donkey anti-mouse (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA).