Rabbit anti p p38

Immunostaining was conducted as previously described [7 (link)]. Briefly, the whole pancreas was fixed in buffered formalin, embedded in paraffin, cut into sections and stained with guinea pig anti-insulin (1:100 dilution; Abcam) and/or rabbit anti-p-P38 (1:1600 dilution; Cell Signaling Technologies) antibodies. Sections were then mounted on glass cover slips and analysed using a Leica DM5000 epifluorescence microscope with Leica Application Suite software (Leica, Milton Keynes, UK) (see ESM Methods).

Western blotting of proteins was performed as described in published protocols (18 (link)). The cell lysates or immunoprecipitated proteins were analyzed by SDS-PAGE. After completion of electrophoresis, the proteins were transferred to a polyvinylidene fluoride or polyvinylidene difluoride (PVDF) membrane, followed by blocking at 4°C for a minimum of 1 h. The blots were probed overnight at 4°C with the primary antibodies. The antibodies used were as follows: mouse anti-Ago2 (Abnova), 1:1,000; rabbit anti-DICER1 (Bethyl), 1:8,000; rat anti-HA (Roche), 1:1,000; horseradish peroxidase (HRP)-conjugated anti-β-Actin (Sigma), 1:10000; rabbit anti-TRBP2 (Cell Signalling), 1:1000; rabbit anti-Drosha (Bethyl), 1:8,000; rabbit anti-P-p38 (Cell Signaling), 1:1,000; rabbit anti-P-ERK1/2 (Cell Signaling), 1:1,000; mouse anti-HSP70 (Santa Cruz Biotechnology), 1:1,000; rabbit anti-MSK1 (Cell Signaling), 1:1,000; mouse anti-HSP70 (Santa Cruz Biotechnology), 1:1,000; rabbit anti-cleaved PARP (Cell Signaling), 1:1,000; rabbit anti-cleaved caspase 9 (Cell Signaling), 1:1,000; and rabbit anti-P-Akt (Ser-473) (Cell Signaling), 1:1,000. Visualization of all Western blots was performed using an UVP BioImager 600 system equipped with VisionWorks Life Science software (UVP) V6.80. ImageJ software was used for densitometric analysis of blots for the relative quantification of bands.

Amlexanox was purchased from Tokyo Chemical Industry (Tokyo, Japan). STAT3 activation inhibitor SPI was purchased from BioVision (Milpitas, CA, USA). LPS from Escherichia coli O111:B4 was obtained from Sigma–Aldrich (St. Louis, MO, USA). The following primary antibodies, which were diluted at 1:1000 ratios in EveryBlot Blocking Buffer (Bio-Rad Laboratories), were used for Western blotting (WB): rabbit anti-COX2 (Cell Signaling Technology), mouse anti-iNOS (Cell Signaling Technology), rabbit anti-p-AKT (Ser473, Cell Signaling Technology), rabbit anti-AKT (Cell Signaling Technology), rabbit anti- IκBα (Cell Signaling Technology), rabbit anti-p- IκBα (Ser32, Cell Signaling Technology), rabbit anti-IKKε (Cell Signaling Technology), rabbit anti-ERK (Cell Signaling Technology), rabbit anti-p-ERK (Thr202/Tyr204, Cell Signaling Technology), rabbit anti-STAT3 (Cell Signaling Technology), rabbit anti-p-STAT3 (Tyr705, Cell Signaling Technology), rabbit anti-NF-κB p65 (Cell Signaling Technology), rabbit anti-p-NF-κB p65 (Ser536, Cell Signaling Technology), rabbit anti-JNK, rabbit anti-p-JNK (Thr183/Tyr185, Cell Signaling Technology), rabbit anti-p38 (Cell Signaling Technology), and rabbit anti-p-p38 (Thr180/Tyr182, Cell Signaling Technology), and mouse anti-β-actin (Santa Cruz Biotechnology). Other chemicals for Western blotting were obtained from Bio-Rad Laboratories.

The jejunum and colon tissues of the mice were fixed in 4% paraformaldehyde for 24 h and embedded in paraffin. Hematoxylin and eosin (H&E) staining was used to stain the jejunum, and images were obtained using a DM3000 microscope (PHASE CONTRAST, Japan). Image-Pro software (ipwin32 software; Media Cybernetics, United States) was used to measure the villous height and crypt depth, and the ratio of villous height to crypt depth was then calculated (Wang et al., 2017 (link)).
For immunohistochemistry (IHC), 5-μm-thick sections of the jejunum and colon tissues were embedded in paraffin, incubated in sodium citrate buffer (pH = 6.0) to repair the antigens and placed in a 3% hydrogen peroxide solution to block endogenous peroxidase after dewaxing and rehydration. To block nonspecific binding sites, the sections were incubated with 3% BSA for 30 min and then incubated with rabbit anti-p-p38 (1:200 dilution, #4511, Cell Signaling Technology), rabbit anti-p-pERK (1:200 dilution, #4370, Cell Signaling Technology), and rabbit and anti-p-pJNK antibodies (1:200 dilution, #4668, Cell Signaling Technology) overnight at 4°C. Then, the sections were incubated with HRP-conjugated secondary antibodies (1:200 dilution, #7074, Cell Signaling Technology) for 50 min and counterstained with hematoxylin after development with DAB buffer (Zhang et al., 2019 (link)).

Whole cochlear pooled protein extracts were prepared from 3 mice, as described [35 (link)]. A fixed volume of extract was separated by SDS-PAGE and transferred to PVDF membranes (0.2 μm, Bio-Rad Laboratories, Hercules, CA, USA) using the Bio-Rad Trans Blot TURBO apparatus. Prior to antibody incubation (overnight 4 °C), membranes were blocked with 5% bovine serum albumin or non-fat dried milk in 0.1% Tween, 1 mM TBS. Primary antibodies used were as follows: rabbit anti-P-p38 (1:1000, #9211), anti-P-JNK (1:1000, #4668), anti-P-AKT (1:1000, #9271) and anti-P-H2AX (1:1000, #2577) (all from Cell Signaling Technology, Danvers, MA, USA), rabbit anti-P22phox (1:250, #sc-20781; Santa Cruz Biotechnology, Dallas, TX, USA), anti-HO-1 (1:1000, #AB1284; Merck, Darmstadt, Germany), anti-MnSOD (1:1000, #06-984; Millipore, Merck, Darmstadt, Germany), goat anti-NQO1 (1:1000, #ab2346; Abcam) and rabbit anti-PI3K (1:15000, in-house). Immunocomplexes were visualized with peroxidase-conjugated secondary antibodies (1 h at room temperature), and bands were detected using the Clarity™ Western ECL Substrate (Bio-Rad) on an Image Quant LAS4000 mini digital camera (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA). Band densities were quantified in triplicate using Image Quant TL software 8.1 (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA ).