Anti p jnk jnk

The lung tissues were homogenized using a homogenizer with RIPA buffer containing a protease inhibitor cocktail (Roche, Indianapolis, IN, USA) and phosphatase inhibitor PhosSTOP (Roche). Total protein was separated by SDS-PAGE and transferred to PVDF membranes (Millipore, Billerica, MA, USA). Membranes were blocked in 5% nonfat milk and then processed with primary antibodies: anti-p-ERK/ERK (1 : 1000), anti-p-JNK/JNK (1 : 1000), anti-p-P38/P38 (1 : 1000), anti-eotaxin (1 : 1000), and anti-IL-25 (1 : 1000) (Cell Signaling Technology Inc., Beverly, MA, USA); anti-galectin-1 (1 : 200) and anti-EPX (1 : 200) (Santa Cruz Biotechnology, CA, USA); and anti-GAPDH (1 : 5000) (KANGCHEN Biotech, Shanghai, China). All of the membranes were subsequently incubated with HRP-conjugated anti-rabbit IgG (Promega, Madison, WI, USA) and polyclonal rabbit anti-mouse IgG (Dako, Copenhagen, Denmark), and all of the blots were detected via enhanced chemiluminescence (ECL; Thermo Scientific). For quantitative analysis, the intensity of protein bands was determined using ImageJ 1.38x software (NIH, Bethesda, MD, USA).

Tissue was rapidly dissected from HECTD1^f/f or wild type mice. Cells were treated with Nuclear and Cytoplasmic Extraction Kit (Beyotime, #P0028). Proteins were extracted in a RIPA lysis buffer (Beyotime, #P0013B), separated via sodium dodecyl sulfate–polyacrylamide gel electrophoresis and electrophoretically transferred onto polyvinylidene fluoride membranes. After blocking with 5% nonfat dry milk in Tris-buffered saline with Tween-20 (Aladdin, #T104863), the membranes were probed with antibodies overnight at 4 °C, followed by incubation with a horseradish peroxidase-conjugated goat anti-mouse (ZSGB-BIO, #ZB5305) or goat anti-rabbit (ZSGB-BIO, #ZB5301) IgG secondary antibody (1:2000). The antibodies were as follows: anti-GFAP (#G3893), obtained from Sigma-Aldrich; anti-σ-1R (#sc137075), purchased from Santa Cruz Biotechnology; anti-GAPDH (#60,004), anti-HECTD1 (#20,605–1-AP), obtained from Proteintech Group; anti-Histone H3 (#9715S), anti-p-ERK/ERK (#9106S/9107S), anti-p-JNK/JNK (#9251S/9252S), anti-p-Akt/Akt (#9271S/9272S), anti-p-p38/p38 (#9211S/9212S), obtained from Cell Signaling Technology; anti-FOXJ2 (#36,867), purchased from Signalway Antibody LLC. Detection was performed using a MicroChemi 4.2® (DNR, Israel) digital image scanner. The band intensity was quantified using ImageJ software (NIH).