Goat anti rabbit igg horseradish peroxidase conjugate

HPASMCs (ScienCell Research Laboratories, Carlsbad, CA, USA) were serum-starved overnight and treated with recombinant human PDGF-BB or H
₂O
₂ for 10, 15 or 30 min. Protein thiol states were monitored using SulfoBiotics Protein Redox State Monitoring Kit Plus (Dojindo Molecular Technologies, Rockville, MD, USA) in accordance with the manufacturer’s instructions. Briefly, cells were washed, proteins precipitated with trichloroacetic acid and “Protein-SHifters” were added to each sample. Samples were then loaded onto a sodium dodecyl sulfate polyacrylamide gel and electrophoresed. The gel was exposed to UV light to cut the “Protein-SHifters.” The resultant non-reducing SDS polyacrylamide gel was electroblotted to a nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA, USA). The membrane was blocked with 5% milk for 30 min at room temperature and incubated with the anti-Prx6 antibody produced in rabbit (Sigma-Aldrich Chemical Company, St. Louis, MO, USA; Catalogue no. P0058; 1:1,000 dilution) at 4°C overnight. The membrane was then washed three times and incubated with goat anti-rabbit IgG-horseradish peroxidase conjugate (Bio-Rad; Catalogue no. 1706515; 1:3,000 dilution) for 45 min at room temperature. After washing three times, signals were obtained using an Enhanced Chemiluminescence System (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA).

For detecting the presence of GFP protein in transgenic lines, the total proteins were first extracted from mature leaf tissues using the Plant Total Protein Extraction kit (Sigma-Aldrich). The protein extract was then subjected to SDS-PAGE. They were heated with NuPAGE^TM LDS sample buffer containing 10% reducing agent (500 mM DTT) at 70°C for 10 min, then analyzed under a NuPAGE^TM 4–12% Bis-Tris gel with a MES SDS running buffer containing antioxidant as instructed by the manufacturer. For immunoblotting, proteins were transferred onto a PVDF membrane (Bio-Rad, Hercules, CA, United States). The membrane was then blocked at 25°C for 1 h with 15% (w/v) dry milk dissolved in PBST. It was then incubated at 25°C for 1 h with 1:200 diluted primary antibody anti-GFP (B-2) (sc-9996; Santa Cruz Biotechnology, Dallas, TX, United States) followed by incubation in the secondary antibody Goat anti-Rabbit IgG horseradish peroxidase conjugate (Bio-Rad). The luminescent signals were generated after incubation with SuperSignal^® West Pico Chemiluminescent substrate (Pierce biotechnology, Rockford, IL, United States) and captured by Kodak Biomax X-ray film (PerkinElmer, Waltham, MA, United States). For staining the PVDF membrane, 0.2% (w/v) Amido Black 10B (MP Biomedicals, Santa Ana, CA, United States) in 10% (v/v) acidic acid was used.

Surface flagella were harvested as previously described [52] (link). Briefly, P. aeruginosa strains were grown at 37°C in LB (with antibiotics as required), harvested by centrifugation, and resuspended in 50 mM sodium phosphate (pH 7.0) containing 10 mM MgCl₂. Flagella were sheared by blending bacterial suspensions for 20 seconds in a Waring blender. Flagellar detachment was monitored by viewing cells under the microscope for loss of swimming. Samples were centrifuged at 12,000×g for 30 min at 4°C to pellet cell bodies. The supernatants containing sheared flagella were separated by SDS-PAGE and blotted to PVDF. Flagellin was detected using anti-FliC antiserum (1∶4000) [53] (link). A goat antirabbit IgG horseradish peroxidase conjugate was used as a secondary antibody (1∶4000, Bio-Rad). Detection was performed using ECL substrate (100 mM Tris-HCL pH 8.5, 250 mM luminol, 90 mM coumarate, 0.009% H₂O₂), and blots were visualized and anlysed using an Image Station 2000R running Carestream MI Software Version 5.0.2 (Kodak). Protein in cell pellets was determined by BCA assay (Pierce) and supernatant equivalent to 25 µg was loaded on the gel for analysis. Rabbit polyclonal antiserum generated against P. aeruginosa Hfq was used to confirm equal loading of protein samples.