Gelpro v3

A total of 30 μg cell lysates or nuclear extracts was resolved on 8%‐12% SDS‐PAGE and examined by Western blot analysis as described.12 β‐Actin was an internal control. All immunoblotting analyses were repeated at least three times with similar results. The protein levels were quantified by densitometry (Gel Pro v.3.1, Media Cybernetics).

Skin tissues were homogenized using a tissue extraction reagent, centrifuged at 10, 000 × g for 20 min, and the supernatants were collected. HaCaT cells were washed with 1X PBS and then lysed with RIPA lysis buffer containing the protease and phosphatase inhibitors (Atto, Tokyo). After sonication, the cell lysates were centrifuged at 8000  × g for 15 min, and the supernatants were collected. The total protein levels were determined using the Bradford protein assay reagent (BioRad, CA, USA). Subsequently, 25–50 μg of the total proteins were separated by 7.5–10% SDS-PAGE electrophoresis and transferred to PVDF membranes (Merck Millipore, Darmstadt, Germany). The proteins were then blocked with 5% skim milk in 1X PBS for 2 h at room temperature. They were then incubated with the primary antibodies, followed by the secondary antibody horseradish peroxidase-conjugated anti-IgG. The blots were detected by enhanced chemiluminescence (BioRad), and the band intensities of the proteins were quantified using GelPro V3.1 software (Media Cybernetics, MD, USA).

ATPase activity of LF was analyzed as in [19 (link)]. Reaction mixtures (10–20 µL) containing 50 mM Tris-HCl, pH 6.8, 1.0 mM MgCl₂, 0.3 mM ATP, 5 µM LF were incubated for 48 h at 37 °C. The ATP hydrolysis products were analyzed by thin-layer chromatography in 0.25 M KH₂PO₄ buffer, pH 7.0, on fluorescent PEI-cellulose plates (Merck). The plates were dried: The positions of adenosine-5’-dophosphate (ADP) were identified using its absorption (dark spots on a uniformly fluorescent plate background). The intensity of the spots of the initial ATP and product of its hydrolysis ADP was evaluated using scanning and then quantified by GelPro v3.1 software (Media Cybernetics, Bethesda, MD, USA). At first, ATPase activity was evaluated from the transition of ATP to ADP (%), and then recalculated to µmol ATP/1 h/1 mg LF taking into account the initial concentration of ATP (0.3 mM) and LF.

Skin tissues were homogenized with an ice-cold tissue extraction reagent, centrifuged at 10,000 ×g for 20 min, and supernatants were collected. On the other hand, HaCaT cells were washed with ice-cold 1X PBS, lysed with RIPA lysis buffer containing protease and phosphatase inhibitors (Atto, Tokyo, Japan), and lysates were centrifuged at 8000 ×g for 15 min, and supernatants were collected. Amounts of proteins in samples were determined using Bradford protein assay reagent (BioRad, CA, USA). Proteins (25–50 µg) were separated by 10–12% SDS-PAGE electrophoresis and transferred to polyvinylidene difluoride membranes (Merck Millipore, Carrigtwohill, Ireland), which were blocked with 5% skim milk in 1X PBS for 2 h at room temperature and incubated with the primary antibodies followed by secondary antibody horseradish peroxidase-conjugated anti-IgG. Proteins were detected by enhanced chemiluminescence (BioRad, CA, USA), and band intensities were quantified using GelPro V3.1 software (Media Cybernetics, MD, USA).

The reaction mixtures (10–30 μL) for analysis of MBP- and histone-hydrolyzing activities of IgGs contained 20 mM Tris-HCl (pH 7.5), 0.7–1.0 mg/mL MBP or mixture of histones, and 0.01–0.2 mg/mL of IgGs. They were incubated for 1–21 h at 37 °C. The cleavage products of the proteins were analyzed after SDS-PAGE using 12% or 4–15% gradient gels; proteins were stained using Coomassie R250. The gels were scanned and quantified by GelPro v3.1 software (Media Cybernetics, Silver Spring, MD, USA). The relative activities (RAs) of different IgGs were determined from a decrease in the percentage of non-hydrolyzed proteins converted to their different shorted forms, taking into account percentage of the hydrolysis of control MBP or histones without Abs. All initial rates (% of the hydrolysis) were estimated using the conditions of the pseudo-first order reaction within the linear regions of the IgG concentrations and time course (20–40% hydrolysis of the proteins). Finally, relative protease activity was recalculated as nmole MBP/1 mg of IgGs/1 h. Protease activity of IgGs was also determined in the same way after isoelectrofocusing of IgGs.

BV2 cells were lysed using the EzRIPA lysis kit (Atto, Tokyo, Japan). Proteins were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes (Merck Millipore, Carrigtwohill, Ireland). Membranes were blocked with 5% skim milk and incubated with primary and secondary antibodies. The primary antibodies used included COX-2 (1:1,500, Santa Cruz Biotechnology, United States) and β-actin (1:10,000, Sigma-Aldrich). Protein bands were developed using enhanced chemiluminescence reagents and visualized using a ChemiDoc imager (Bio-Rad Laboratories). Band intensities were measured using GelPro v.3.1 software (Media Cybernetics, Rockville, MD, United States).

HaCaT cells were lysed with a RIPA lysis buffer, containing protease and phosphatase inhibitors (Atto, Tokyo, Japan). After sonication, the cell lysates were centrifuged at 8000 x g for 10 min, and the supernatants were collected. The protein concentration was determined using a Bradford protein assay reagent (Bio-Rad, CA, USA). Subsequently, 30–50 µg of the total proteins were separated by 5–10% SDS-PAGE electrophoresis and transferred to polyvinylidene difluoride membranes (Merck Millipore, Carrigtwohill, Ireland). After blocking for two hours in 5% skim milk in 1X PBS at room temperature, the membranes were incubated with the primary antibodies followed by the secondary antibody horseradish peroxidase-conjugated anti-IgG. All membranes were detected by enhanced chemiluminescence (Bio-Rad, CA, USA). The band intensities of the proteins were quantified using the GelPro V3.1 software (Media Cybernetics, MD, USA).