Enhanced chemiluminescence detection reagent

Liver tissue samples were solubilized in radioimmunoprecipitation assay buffer containing protease inhibitors (Pierce, Rockford, IL, USA). The lysates were centrifuged (13,000× g for 10 min at 4 °C), and supernatants were boiled with sodium dodecyl sulfate buffer (0.5 M β-mercaptoethanol). Later, the lysates were subjected to 12% SDS–PAGE for Western blot analysis. Reactive protein bands were analyzed by enhanced chemiluminescence detection reagent (Amersham Biosciences, Piscataway, NJ, USA), using an image reader LAS-3000 (version 2.1; Fujifilm, Tokyo, Japan). HNF4α polyclonal antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) were diluted 1:500 in TBS-T/5% BSA. Horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG antibody diluted 1:2000 (Santa Cruz Biotechnology, Inc.) in TBS-T/5% BSA was used as a secondary antibody. The experiment was repeated using three different samples.

Tissues were lysed using radioimmunoprecipitation assay (RIPA) lysis buffer containing a mixture of protease inhibitors (Sigma-Aldrich, St. Louis, MO, USA). Adenoid protein samples (30 μg) were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. The membrane was first incubated with primary antibodies against IL-17C and GAPDH (Abcam, Cambridge, UK), washed, and then incubated with secondary antibodies, namely horseradish peroxidase-labeled goat anti-rabbit or goat anti-mouse immunoglobulin (Jackson Labs, Bar Harbor, ME, USA). Signals were detected using the Enhanced Chemiluminescence detection reagent (Amersham, Buckinghamshire, UK) and relative band intensities were calculated using the Image J software (National Institutes of Health, Bethesda, MD, USA). The total number of adenoid tissues analyzed in each western blot assay is indicated in the corresponding figure legend.

Cells were lysed using RIPA buffer (Biosolution, Korea), phenylmethylsulfonyl fluoride (Sigma), protease inhibitor, and phosphatase inhibitor cocktail 2/3 (Sigma) and then centrifuged at 25,000 x g for 30 min. Cell extracts were quantified using the BCA protein assay kit (Pierce Biotechnology, USA) according to the manufacturer's instructions. The proteins (20-30 μg) were separated on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride membranes (GE Healthcare, UK) for western blot analyses. The transferred membrane was blocked with 1x Tris-buffered saline with Tween 20 (Sigma) (TBST) containing 5% skim milk (BD Biosciences) for 1 h and then incubated with primary antibodies ADAM10 (Santa Cruz), ADAM17 (Abcam, USA), β-arrestin (Bethyl, USA), and CXCR6 (GeneTex) in 1x TBST containing 1% skim milk at 4°C overnight. The membrane was washed three times with 1x TBST for 10 min, then incubated with secondary anti-rabbit (Cell Signaling) and anti-mouse (Santa Cruz) antibodies in 1x TBST containing 1% skim milk for 45 min. The membrane was washed three times with 1x TBST for 15 min and was visualized with enhanced chemiluminescence detection reagent (Amersham Pharmacia Biotech, USA) and imaged by ChemiDoc (Bio-Rad Laboratories, USA).

Western blotting was performed as previously described [40 (link)]. Briefly, the protein concentrations in cell lysates and tissue homogenates were measured using a Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA, USA). Equal amounts (20–30 μg) of protein were subjected to SDS-PAGE on 4%–12% Bolt^TM Mini Gels (Novex, Thermo Fisher Scientific, Waltham, MA, USA) and transferred to a Polyvinylidene fluoride (PVDF) membrane. Prestained protein ladders (GangNamstain^TM, iNtRON Biotechnology, Burlington, NJ, USA) covering a broad range of molecular weights were used to detect the molecular weights of the proteins. The membranes were blocked in 5% skim milk/bovine serum albumin (BSA) to reduce nonspecific binding and incubated with primary antibodies (1:1000 dilution) at 4 °C overnight. After reaction with a horseradish peroxidase-conjugated secondary antibody as appropriate, the proteins were detected using an enhanced chemiluminescence detection reagent (Amersham Pharmacia Biotech, Uppsala, Sweden) according to the manufacturer’s instructions. The x-ray films were scanned, and the optical densities of the bands were analyzed through densitometry using the computer-based Sigma Gel program, version 1.0 (SPSS Inc., Chicago, IL, USA). Density values were calculated in arbitrary units (AU) relative to the untreated control.

For determination of the protein expression of STAT-3 and RANKL, the western blot method was employed (Mansour et al., 2021 (link)). Phosphate buffered saline was utilized for the homogenization of the obtained hind limb tissues. Followed by using SDS polyacrylamide gel electrophoresis for the separation of exactly 10 μg protein from each limb sample that are then moved to a nitrocellulose membrane. The obtained membrane was incubated with either anti-RANKL (cat# MBS8813030) or anti-STAT-3 (cat# MBS8808638) antibodies (MybioSource, CA, USA) for 24 hours at 4 °C and the formed blots were examined using enhanced chemiluminescence detection reagent (Amersham Biosciences, IL, USA). The obtained outputs were conveyed as arbitrary units against β-actin (cat# MBS448085) employing image analysis software (Image J, version 1.46a, NIH, Bethesda, MD, USA).