Horseradish peroxidase conjugated goat anti mouse igg secondary antibody

Wild‐type and mutant (G306R) plasmids were transiently transfected into HEK293T (human embryonic kidney) cells cultured in Dulbecco's Modified Eagle's Medium (DMEM), using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol. Total protein extracts (20 μg) from cell lysates were electrophoretically separated on 10% denaturing polyacrylamide gels and then transferred to Polyvinylidene difluoride (PVDF) membranes (Roche, Basilea, Switzerland). A high range SigmaMarker (Sigma‐Aldrich, Saint Louis, MO; molecular weight range, 36 000‐200 000 kDa) was used to characterize protein migration. Membranes were blocked in Tris‐buffered saline with 0.1% Tween 20 containing 5% non‐fat dried milk and reacted overnight at 4°C with the primary monoclonal antibody, anti‐FLAG mouse M2 clone (Sigma‐Aldrich) diluted 1:2000, and anti‐beta Actin antibody ab8226 (Abcam, Cambridge, UK), diluted 1:1000. After rinsing, filters were incubated with horseradish peroxidase‐conjugated goat anti‐mouse IgG secondary antibody (Santa Cruz), diluted 1:10000. Signals were developed using the enhanced chemiluminescent horseradish peroxidase (HRP) substrate Westar ETA C ultra (Cyanagen, Bologna, Italy), and specific protein bands were revealed using the G:BOX Chemi XT4 gel doc system (Syngene, Frederick, MD).

Normal human epidermal keratinocytes were seeded into 12-well plates and cultured as described above. Cells were collected and disrupted in lysis buffer (Cell Signaling Technology, Boston, MA, United States). After adding SDS sample buffer (Cell Signaling Technology), lysates were electrophoretically separated on a 12% polyacrylamide gel (ATTO Corp., Tokyo, Japan). Proteins were electrophoretically transferred onto a polyvinylidene difluoride membrane (Bio-Rad, Hercules, CA, United States). The membrane was blocked in 5% non-fat dry milk in Tris–buffered saline (TBS) with 0.1% Tween-20 (TBST) for 1 h at room temperature. After several washes with TBST, the membrane was incubated overnight at 4°C with primary mouse anti-human IL-36 beta antibody (R and D system; 1:1000), anti-human IL-36 gamma antibody (R and D system; 1:1000), anti-human p19 antibody (Proteintech, Tokyo; 1:1000), or anti-human tublin antibody (Proteintech; 1:1000). The membrane was washed several times in TBST followed by a 1-h incubation with horseradish peroxidase–conjugated goat anti-mouse IgG secondary antibody (Santa Cruz, Dallas, TX, United States).

RAW 264.7 cells (1 × 10⁶ cells/mL) were preincubated for 16 h and then treated with LPS (1 μg/mL). After incubation for 24 h, the cells were harvested and washed twice with phosphate-buffered saline. The cell lysates were prepared in RIPA buffer (50 mM/L Tris–HCl [pH 7.4], 150 mM/L NaCl, 1% Triton X-100, 0.1% sodium dodecyl sulfate (SDS), and 1 mM/L EDTA; Thermo Fisher Scientific, MA, USA) for 30 min on ice. The cell lysates were centrifuged at 13,000 × g for 15 min at 4 °C, and 30 μg of the cell lysate was separated by to 7.5% SDS-polyacrylamide gel electrophoresis. The separated proteins were transferred onto a PVDF membrane (Bio-Rad, CA, USA). Nonspecific binding to the membrane was blocked by incubation with blocking buffer (Thermo Fisher Scientific) for 60 min at room temperature. Then, the membrane was incubated with anti-mouse iNOS (1 : 500; Santa Cruz, CA, USA) and anti-mouse COX-2 (1 : 1000; BD Biosciences Pharmingen, CA, USA) overnight at 4°C. After washing, the blots were incubated with horseradish peroxidase-conjugated goat anti-mouse IgG secondary antibody (1 : 1000; Santa Cruz) for 60 min at room temperature. The bands were visualized by using the LAS-4000 luminescent image analyzer (GE Healthcare Life Sciences, NJ, USA) and ECL detection reagent (GE Healthcare Life Sciences).

Three pairs of individual WUS samples from the same set used in the ELISA analysis were randomly selected and used for immunoblotting analysis. The protein concentration was determined using the BCA™ protein assay kit (Pierce, Rockville, IL, USA), according to the manufacturer’s protocol. Five μg of total protein in each individual sample was separated by 8% or 12% SDS-PAGE. The separated proteins were transferred to a nitrocellulose membrane and subsequently probed with one of the following antibodies: polyclonal rabbit anti-human C3c complement antibody (DAKO, Glostrup, Denmark); polyclonal goat anti-fibrinogen β antibody (Santa Cruz Biotechnology, Inc.USA,); monoclonal mouse anti-cystatin SA antibody (Abcam, Cambridge, UK). Subsequently, the membranes were incubated with one of the following antibodies: for C3c, horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody (Bio-Rad Laboratories, Inc.USA ,); for fibrinogen β, horseradish peroxidase-conjugated donkey anti-goat IgG secondary antibody (Bio-Rad Laboratories, Inc., USA); for cystatin SA, horseradish peroxidase-conjugated goat anti-mouse IgG secondary antibody (Santa Cruz Biotechnology, Inc., USA). Immunoreactive protein bands were visualized using Western Lightning® Plus-ECL substrate (PerkinElmer, Inc., Waltham, MA, USA).

A375 cells were seeded onto 6-well plates and cultured as described above. Cells were collected and disrupted in lysis buffer (Cell Signaling Technology, Boston, MA, USA). After adding SDS sample buffer (Cell Signaling Technology), lysates were electrophoretically separated on a 12% polyacrylamide gel (ATTO Corp., Tokyo, Japan). Proteins were electrophoretically transferred onto a polyvinylidene difluoride membrane (Bio-Rad, Hercules, CA, USA). The membrane was blocked in 5% nonfat dry milk in Tris-buffered saline (TBS) with 0.1% Tween-20 (TBST) for 1 h at room temperature. After several washes with TBST, the membrane was incubated overnight at 4 °C with primary mouse anti-human IL-23p19 antibody (Lifespan Bioscience, 1:1000) or mouse anti-human beta-actin antibody (Cell Signaling Technology, Tokyo, Japan; 1:1000). The membrane was washed several times in TBST followed by 1 h incubation with horseradish peroxidase–conjugated goat anti-mouse IgG secondary antibody (Santa Cruz, CA, USA).