Rabbit anti human p38

Confluent cell monolayers were harvested using cell lysis buffer^® (Cell Signaling Technology) according to the manufacturer’s instructions. Nuclear and cytoplasmic extracts were separated using NE-PER nuclear and cytoplasmic extraction reagents (Pierce Biotechnology, Rockford, IL), respectively. Whole cell lysates, as well as nuclear and cytoplasmic fractions were separated by SDS-PAGE, and transferred to polyvinylidene difluoride membranes (ATTO, Tokyo, Japan) using a semidry transblot system (Bio-Rad Laboratories, Hercules, CA). Non-specific binding on the blots was blocked with 0.5% (w/v) non-fat dried milk and 0.1% (v/v) Tween 20 in PBS overnight at 4°C, followed by incubation for 1 h at RT with rabbit anti-human IL-33 mAb (EPITOMICS), mouse anti-human GAPDH mAb (MBL, Tokyo, Japan), rabbit anti-human p38 (Cell Signaling Technology), or with p38 polyclonal antibodies (Cell Signaling Technology) at 1:1,000 for 1 h at RT. Blots were incubated with HRP-conjugated goat anti-rabbit IgG (Cell Signaling Technology) at 1:2,000 for 1 h at RT and then soaked in Immunostar^® (Wako Pure Chemical Industries). Chemiluminescent signals were detected using an LAS-4000IR luminescent image analyzer (Fujifilm, Tokyo, Japan). The intensity of expression was quantified by densitometry using ImageJ software.

The proteins of DVSMCs were extracted using RIPA lysis buffer (Beyotime), with the protease inhibitor phenylmethanesulfonyl fluoride (Beyotime). The proteins were separated into 8% or 10% SDS-PAGE (Beyotime), and then transferred onto PVDF membranes (EMD Millipore, Billerica, MA, USA). After being blocked with 5% milk for 2 h at room temperature, the membranes were incubated using primary antibodies, including rabbit anti-human collagen 1 (1:1000, Abcam), rabbit anti-human collagen 3 (1:3000, Abcam), rabbit anti-human α-SMA (1:600, Abcam), goat anti-human IL-17RA (1:1000, Abcam), rabbit anti-human p38 (1:1000, Cell Signaling Technology, Boston, MA, USA), rabbit anti-human phospho-p38 MAPK (1:1000, Cell Signaling Technology), rabbit anti-human JNK (1:1000, Cell Signaling Technology), rabbit anti-human phospho-JNK (1:1000, Cell Signaling Technology), rabbit anti-human ERK1/2 (1:1000, Cell Signaling Technology), rabbit anti-human phospho-ERK1/2 (1:1000, Cell Signaling Technology), and rabbit anti-human GAPDH (1:1000, Cell Signaling Technology) overnight at 4°C. The membranes were then washed and incubated with appropriate HRP-conjugated secondary antibodies for 1.5 h at room temperature. The proteins were detected using ECL detection reagents (Beyotime).