Phospho jnk

The expression of proteins of interest in BMDCs was analyzed using western blotting as previously described 1 (link). The following primary antibodies were used: anti-TGR5 (Abcam, Cambridge, MA, USA), FXR (Abcam), Bax (Abcam), BCL-2 (Abcam), caspase3 (Abcam), cleaved-caspase3 (Abcam), LC3 (Abcam), Belin1(Abcam), P62 (Abcam), P65 (Abcam), Ikbα (Abcam), ERK1/2 (Santa Cruz), P38 (Abcam), JNK (Abcam), phospho-P65 (CST), phospho-Ikbα (Abcam), phospho-ERK1/2 (Santa Cruz), phospho-P38 (Abcam), phospho-JNK (Abcam), and β-actin (Abcam).

The total proteins of tissues and cell samples were extracted using a radioimmunoprecipitation assay buffer (Solarbio. Equal amounts of protein (20 μg) were separated by sodium dodecyl sulfate–, Beijing, China)polyacrylamide gel electrophoresis and then transferred to polyvinylidene fluoride membranes (Millipore, MA, USA). After blocking with 5% bovine serum in TBST, the membranes and corresponding primary antibodies were incubated overnight in a shaker at 4°C. The primary antibodies used were as follows: anti-GAPDH (1:10,000, ProteinTech, Wuhan, China), anti-HSF2BP (1:1000, Abcam, USA), ERK (1:500, Abcam, USA), phospho-ERK (1:500, Abcam, USA), JNK (1:1,000, Abcam, USA), phospho-JNK (1:1,000, Abcam, USA), p38 (1:1,000, Abcam, USA), and phospho-p38 (1:1,000, Abcam, USA). After incubation with horseradish peroxidase-conjugated secondary antibodies (1:10,000, ProteinTech, Wuhan, China) at room temperature for 1 h, the membranes were visualized using the enhanced chemiluminescence method (Thermo, MA, USA) with a chemiluminescent detection system (Tanon, Shanghai, China).

Right lung (40 mg) was cut off. Then, 400 μL of CER I and 4 μL of Halt™ Protease and Phosphatase Inhibitor Cocktail (Pierce Biotechnology Inc., Rockford, USA) homogenate were added to the lung tissue. The mixture was incubated on ice for 10 min followed by addition of precooled (4°C) CER II (22 μL). The supernatant was collected by centrifugation for 10 min at 16000 rpm. Protein centration was measured by BCA method. Equal amounts of proteins were loaded in a 12% SDS-PAGE gel. After blotting onto PVDF membrane, samples were incubated with respective primary antibodies: phospho-ERK, C-jun (Cell Signaling Technology, Boston, MA), phospho-p38, phospho-JNK, ERK, p38, JNK, C-fos, γ-GCS-h (Abcam, Cambridge, UK), and β-actin (1 : 1000 dilution) and were incubated at 4°C overnight. Then, the respective secondary antibodies conjugated to HRP were incubated for 1 h, followed by three-time washing. The membrane was then incubated with ECL (Millipore, MA, USA) for luminescence generation. The image and the gray level were analyzed by Alpha Innotech and Adobe software. Protein expression level was evaluated by the β-actin ratio.