Ecl plu

IL‐38 expression in murine CD4⁺CD25⁺ Tregs was measured by Western blot following the manufacturer's instructions. In brief, nitrocellulose membranes were incubated at 4°C overnight with polyclonal rabbit antibodies against IL‐38 (1:1000) and monoclonal mouse antibody against β‐actin (1:1000), followed by incubation with horseradish peroxidase‐conjugated polyclonal goat anti‐rabbit secondary antibody (1:5000) or monoclonal rabbit antimouse antibody (1:5000) at room temperature for 1 hour. Finally, Western blot bands were visualized using ECL^Plus (Amersham Biosciences) on an ImageQuant LAS 4000 biomolecular imager (GE Healthcare Life Sciences) and analysed using ImageJ software (US National Institutes of Health, https://imagej.nih.gov/ij/).

Western blot analysis was performed using the left lung [27] (link). Briefly, lung tissue was homogenized, and aliquots of each fraction were used to measure the protein concentration of each sample using a detergent compatible assay (Bio-Rad, Hercules, CA). Equal amounts of protein samples (40 µg) were subjected to electrophoresis in an SDS-PAGE gel and transferred onto a nitrocellulose membrane. Membranes were then blocked with blocking buffer for two hours, followed by incubation with the primary antibodies overnight at 4°C: rabbit polyclonal anti- IL-1β antibody (1∶1000) (Millipore Biosciences Research Reagents SBU, Temecula, CA), mouse monoclonal anti-myeloperoxidase (MPO, 1∶500), rabbit polyclonal anti-ZO-1 (1∶1000) and mouse monoclonal anti-Occludin (1∶1000) (Santa Cruz Biotechnology, Santa Cruz, CA). For loading control, β-Actin (1∶3000) (Santa Cruz Biotechnology, Santa Cruz, CA) was blotted on the same membranes. Immunoblots were processed with appropriate secondary antibodies (1∶2000) (Santa Cruz Biotechnology, Santa Cruz, CA) for one hour under room temperature. Blot bands were detected with a chemiluminescence reagent kit (ECL Plus; Amersham Bioscience, Arlington Heights, IL). The bands were quantified by densitometry with Image J software.

An immunoblotting protocol was used for examining iNOS/PERK/GRP-78/CHOP axis as described [53 (link),54 (link)]. Briefly, homogenization of the hippocampus tissue was applied in a cold lysis buffer (Tris-HCL [50 mmol/L; pH 8.0], SDS [0.1% w/v], NP40 [1% v/v], NaCl [150 mmol/L], sodium deoxycholate [0.5% w/v], and phenylmethylsulfonylfluoride [0.5 mmol/L]). The lysate was centrifuged at 10,000× g and the supernatant was collected. The protein lysates (50 μg/lane) were subjected to SDS-PAGE and the separated proteins were transferred into a nitrocellulose membrane (Bio-Rad, Hercules, CA, USA). Following membrane blocking with non-fat dried milk in TBST, primary antibody incubation was applied overnight at 4 °C using specific antibodies against iNOS (Abcam, Waltham, MA, USA, Cat. # ab 178945), eNOS (Cat. # 32027), CHOP (Cat. # 2895), PERK (Cat. # 3192), and GRP78 (Cat. # 3183) at 1:1,000 dilution (Cell Signalling Technology, Beverly, MA, USA). After washing, the membranes were incubated with HRP-tagged secondary antibody (1:10,000, Cell Signalling Technology, Beverly, MA, USA) for 1 h at room temperature. Finally, protein visualization was performed using an enhanced chemiluminescence kit (ECL plus; Amersham, Arlington Heights, IL, USA) and band intensity quantification was applied using Molecular Analyst Software (Bio-Rad, Hercules, CA, USA).

The aortas were homogenized and lysed. Proteins (50 μg) were separated in precoated SDS-PAGE 4-15% (MiniPROTEAN® TGX™, Bio-Rad), transferred to nitrocellulose membrane, and then incubated (2 h at room temperature) in blocking buffer (5% nonfat dry milk in PBS). The membranes were probed overnight at 4°C with primary rabbit polyclonal antibody directed to arginase-1 (1/200, Santa Cruz Biotechnology). After 3 washes, immunoreactive bands were revealed with a secondary peroxidase-conjugated anti-rabbit IgG (1 : 5000, Beckman Coulter), detected by enhanced chemiluminescence system (ECL Plus, Amersham Biosciences), and quantified by densitometry. A polyclonal anti-α-tubulin antibody (1 : 5000, Sigma-Aldrich) was used to check protein gel loading and to normalize protein expression. The analysis of the blots was performed with Image Lab™ software v4.1 (Bio-Rad).

Protein from MCF7 and MDA-MB-231 parental or mammosphere-derived cells pre-exposed to different treatments, namely, anti-CDH11 antibody, miR-335 mimic, or miR-335 inhibitor, were loaded (20 μg/lane) and separated using 10% sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE), then the blots transferred onto polyvinylidene difluoride (PVDF) membranes, blocked in 5% skim milk in TRIS buffered saline (TBS) with Tween-20 (TBST) for 1 h, at room temperature, then incubated overnight at 4 °C with the primary antibodies against CDH11 (1:1000, clone 2C67, #H00001009-M25), E-cadherin (1:1000, clone 7H12, #MAB16359), vimentin (1:1000, clone 1G3, #H00007431-M10), b-catenin (1:2000, #MAB11143), FYN (1:1000, clone 3G11-F9, #H00002534-M01), EZH2 (1:1000, clone 2C3, #H00002146-M01), and b-actin (1:2000, clone 3G4-F9, #H00000060-M01) all purchased from Abnova (Abnova Corporation, Taipei, Taiwan). This was followed by incubation in peroxidase - conjugated secondary antibody for 1 h at room temperature, and washed with TBST three times. The signals were developed using enhanced chemiluminescence (ECL-Plus, Amersham Pharmacia Biotech, Piscataway, NJ, USA) and detected with a UVP BioSpectrum® Imaging System (Analytik Jena US LLC, Upland, CA, USA).

Seven-day-old WT and aer Arabidopsis seedlings were ground to a powder in liquid nitrogen using a mortar and pestle, and the resulting powder was suspended in the extraction buffer (100 mM Tris-HCl buffer, pH 7.5, containing 0.1mM EDTA, 7% (w/v) PVPP, 5% Suc, 0.0005% Triton X-100, 1 mM PMSF, 15 mM DTT, and a commercial cocktail of protease inhibitors (AEBSF, 1,10-phenantroline, pepstatin A, leupeptine, bestatine, and E-64 from Sigma-Aldrich; 1/2, FW/v). Then, the crude extracts were centrifuged twice at 3,000xg for 6 min. Total protein content was analyzed by Bradford assay and separated by 10% SDS-PAGE and transferred to PVDF membranes (Immobilon P, Millipore, Bedford, MA, USA). For AER immunodetection, an specific antibody against Arabidopsis AER (Mano et al., 2005 (link)) was used at a dilution of 1:1000 and the immunoreactive band was detected using a photographic film (Hyperfilm, Amersham Pharmacia Biotech) with an enhanced chemiluminescence kit (ECL-PLUS, Amersham Pharmacia Biotech).