Proteinase inhibitors cocktail

Tissues were frozen in liquid nitrogen and ground to powder in a frozen state in a ball mill (Retsch, Germany) for 2 min at 30 min⁻¹. Protein was extracted using 50 mm Tris–HCl pH 8.0, 20 mm EDTA, 1 mm DTT and 1% proteinase inhibitors cocktail (Sigma, P9599). Before electrophoresis the samples were heated at 95°C for 5 min in the presence of loading buffer (50 mm Tris–HCl pH 6.8, 2% SDS, 10% glycerol, 100 mm DTT, 12.5 mm EDTA, 0.02% bromophenol blue). The proteins were separated on an 8.5% SDS‐PAGE according to (Laemmli, 1970) and electro‐blotted onto nitrocellulose membrane (Amersham Protran, GE Healthcare Life Science) at 100 V for 45 min. The membrane was blocked with 2.5% skimmed dried milk (in PBST buffer) for 1 h. The citrin tag was detected with anti‐GFP (D5.1) XP^® Rabbit mAb (Cell Signalling; 1:1000 in blocking buffer) followed by horseradish peroxidase‐conjugated anti‐rabbit secondary antibody (Cell signalling; 1:10 000 in PBST) and Amersham ECL Prime Western Blotting Detection Reagent (Amersham, GE Healthcare Life science). Blots were exposed to X‐ray film (Amersham, GE Healthcare Life Science) and recorded on a ChemiDoc XRS Imager (Bio‐Rad). Endo H (New England BioLabs) and PNGase F (New England BioLabs) digestions were performed as previously described (Hüttner et al., 2012).

Total cell extracts were lysed using a RIPA lysis buffer (Beyotime, China) supplemented with proteinase inhibitors cocktail (Sigma, USA). The protein concentration was measured using the BCA assay kit (Sigma, USA). First, 20 μg of protein from total cell lysates was separated by 10% SDS–polyacrylamide gel electrophoresis then transferred to PVDF membranes (Invitrogen, USA). The membranes were then incubated in a blocking buffer containing 5% nonfat dry milk for 1 hr at room temperature and were incubated with the different primary antibodies overnight at 4°C. The membrane samples were then washed with TBS-T 3 times and incubated with the corresponding secondary antibodies for 1 hr at room temperature. We used ECL western blotting substrate (Pierce, USA) to test the immunoblotted bands. The primary antibody preparations were as follows: HIF-1α antibody, SCF antibody, and β-actin antibody.

Tissue samples were put in ice-cold Tissue Protein Extraction Reagent (Pierce, USA) containing a mixture of proteinase inhibitors cocktails (Sigma, USA) and phosphatase inhibitors (Roche, Switzerland) to be homogenated by the tissue homogenizer. Samples were then centrifuged for 30 minutes at 10,000 g. The supernatant was collected, and the protein concentration was measured using the BCA Protein Assay Kit (Pierce, USA). Proteins (80∼120 µg) were separated by SDS–PAGE and subsequently transferred to polyvinylidenefluoride membranes. The membranes were blocked with 5% bovine serum albumin (BSA) in incubation buffer for 2 h at room temperature. Afterwards, they were incubated with the corresponding primary antibodies at 4°C overnight and then with the peroxidase-conjugated secondary antibody at room temperature for 2 h. Finally, the signal was detected by enhanced chemiluminescence. Loading accuracy was evaluated by membrane rehybridization with anti-β-Actin antibodies. ImageJ software was used to quantify western blot signals.

Samples of liver tissues were taken from the hepatic grafts at 6, 24 and 72 h and 7 days after reperfusion in recipients. Parts of them were put in ice-cold Tissue Protein Extraction Reagent (Pierce, USA) containing a mixture of proteinase inhibitors cocktails (Sigma, USA) and phosphatase inhibitors (Roche, Switzerland) to be homogenated by the tissue homogenizer. Samples were then centrifuged for 30 minutes at 10000 g. The supernatant was collected, and the protein concentration was measured using the BCA Protein Assay Kit (Pierce, USA). Proteins (80∼120 µg) were separated by SDS-PAGE and subsequently transferred to polyvinylidenefluoride (PVDF) membranes. The membranes were blocked with 5% bovine serum albumin (BSA) in incubation buffer for 2 h at room temperature. Afterwards, they were incubated with antibodies against p-c-Jun (Epitomics, USA) and CyD1 and GAPDH (Abcam, USA) at 4°C overnight and then with the peroxidase-conjugated secondary antibody at room temperature for 2 h. Finally, the signal was detected by ECL. Loading accuracy was evaluated by membrane rehybridization with anti-GAPDH antibodies. Bands were acquired with a Molecular Imager ChemiDox XRS+ Imaging System with Quantity One Image software and quantified with ImageJ software.