Fusion fx7 spectra

Detection of JNK and p38 activation was performed exactly as described [24 (link)]. In brief, MCs deprived of growth factors were stimulated with IL-33 (20 ng/mL) for 15 min or kept without stimulus (control), then boiled in Laemmli buffer, and lysates resolved by SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis). After transfer to a blotting membrane and incubation with antibodies, proteins were visualized by a chemiluminescence assay (Weststar Ultra 2.0, Cyanagen, Bologna, Italy) and bands recorded on a chemiluminescence imager (Fusion FX7 Spectra, Vilber Lourmat, Eberhardzell, Germany). The following primary antibodies from Cell Signaling Technology (Frankfurt am Main, Germany) were used: Anti-p-p38 (Thr180/Tyr182, #9211), anti-p38 (#9212), anti-p-JNK (T183/Y185, #9251), and anti-JNK (#9252). Bands were quantified by densitometry with the software ImageJ (National Institutes of Health, Bethesda, MD, USA) and the degree of phosphorylation was assessed by the following equation: Ratio of phosphorylated protein = density_{phospo-protein}/density_{total protein}.

Proteins of cochleas were extracted with cell lysis buffer for Western (P0013, Beyotime, China). Protein concentrations were measured with the BCA Protein Assay Kit (P0013, Beyotime, China). Total protein was separated by 8% SDS-PAGE electrophoresis and transferred to PVDF membranes. The membranes were blocked with 5% BSA and incubated with rabbit anti-Dot1l antibody (1:200, ab64077, Abcam, UK) or anti-GAPDH antibody (1:1000, AF1186, Beyotime, China) overnight at 4 ℃ and subsequent HRP-labeled Goat Anti-Rabbit IgG (1:1000, A0208, Beyotime, China) for 1 h at 37 ℃. Immunolabeled bands reacting with a chemiluminescent substrate BeyoECL Star (P0018A, Beyotime, China) were detected and quantified by FUSION-FX7 Spectra (Vilber, France).

All the cell protein was extracted after transfection using RIPA buffer (Solarbio, Beijing, China) added with protease inhibitor cocktail and phosphatase inhibitor cocktail I (MCE, America) after washing the cells with PBS. A BCA kit (Elabscience Bio, Wuhan, China) was then used to quantify the concentration. 20 ug protein from each sample was extracted for electrophoresis on a 10% SDS-PAGE gel and it was transferred onto a polyvinylidene fluoride membrane (PVDF; Millipore, Billerica, MA, USA). Then, the membranes were blocked in 5% fat-free milk for 1 h at 25°C. Next the membranes were washed with TBST three times and incubated at 4°C overnight with primary antibodies, including anti-β-actin (4970T, 1 : 1000, CST), anti-YAP (14074T, 1 : 1000, CST), anti-p-YAP (13008T, 1 : 1000, CST), anti-vimentin (AB-70081, 1 : 1000, Elabscience), anti-E-cadherin (AB-53267,1 : 1000, Elabscience), and anti-zonula occludens-1 (ZO-1) (AB-18170, 1 : 1000, Elabscience). Furthermore, the membranes were washed three times with TBST, which were next incubated at 25°C for 1 h with respective horseradish peroxidase-labeled secondary antibodies. Finally, FUSION FX7 Spectra (Vilber, France) were conducted to acquire the protein bands. Each experiment was independently repeated three times.

To prepare protein samples, liver tissue blocks were pulverized by a high-throughput tissue-grinding apparatus (2 minutes, 1,000 rpm, TL-2020, Ding Hao Yuan Technology Co., Ltd., Beijing, China), and proteins were extracted from the homogenate liver tissue. The nuclear and cytosolic proteins were collected using nuclear and cytoplasmic extraction reagents according to the provided instructions. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to a polyvinylidene fluoride (PVDF) membrane. After being incubated overnight at 4°C with target antibodies against P-/p38 MAPK, P-/JNK, P-/ERK 1/2, NF-κBp65, and IκBα (1 : 1000 dilution for these antibodies), they were then incubated with the goat anti-rabbit IgG. The antibodies β-tubulin (1 : 1000 dilution), histone H3 (1 : 2000 dilution), and GAPDH (1 : 10000 dilution) were used as an internal control. The bands were detected using an enhanced chemiluminescence system (Fusion FX7 Spectra; Vilber Lourmat, Eberhardzell, Germany).

For protein lysates PBECs were treated with RIPA buffer (Pierce) supplemented with 2x Halt Protease and Phosphatase Inhibitor Cocktail (Pierce) and 250 U Benzonase (Sigma) for 15 min on ice. Lysates were centrifuged at 14000 rpm for 15 min at 4°C. Total protein concentration was measured by BCA protein assay (Pierce). 12 μg of protein lysates lysates were mixed with Laemmli buffer (BioRad, Hertfordshire, UK) and 25 mM DTT and boiled for 7 min. Proteins separated on 4%-20% Mini PROTEAN gels (BioRad) and transferred onto nitrocellulose membranes by iBlot (Invitrogen). Membranes were blocked in 5% milk/TBST for 1h and probed with primary antibodies (αbeta-Actin (D6A8, New England Biolabs), αPhospho-Stat1 (Tyr701) (D4A7, New England Biolabs), αStat1 (#9172, New England Biolabs)) at 1:1000 in 5% BSA/TBST over night at 4°C. HRP-linked IgG (New England Biolabs) (1:2000) in 5% BSA/TBST was added the next day for 1h. Membranes were developed with ECL Western blotting substrate (Pierce) and imaged with FUSION FX7 SPECTRA (Vilber).