Se134

Samples (20 μg) were subjected to SDS–PAGE analysis and electrotransferred to membranes (PVDF, IPVH00010, Millipore, USA), washed with TBST, and then incubated with Rabbit Anti-eNOS antibody (1:1000; Bs-20608R, Bioss, China), eNOS (phospho Ser1177) antibody (1:1000; GTX129058, Gene Tex, USA), eNOS (phospho Thr495) antibody (1:1000; PA5–17706, ThermoFisher, USA) or GAPDH Antibody (1:10000; 60,004–1-Ig, Proteintech, China) at 4 °C overnight. After washing with TBST, membranes were incubated with Goat Anti-rabbit IgG/HRP antibody (1:3000; SE134, Solarbio, USA) or Goat Anti-Mouse IgG/HRP antibody (1:3000; SE131, Solarbio, USA). Finally, the results were visualized by an enhanced fluoro-chemiluminescent system (ECL, PE0010, Solarbio, China).

Proteins were extracted by mixing RIPA lysate (R0010, Solarbio) and PMSF (P0100, Solarbio). The concentrations of proteins were assayed by the BCA kit (PC0020, Solarbio). According to different molecular weights, 5% concentrated gel and 8% separation gel concentrations were used in the SDS-PAGE. After transferred to a PVDF membrane, the proteins were blocked by prepared 5% (M/V) BSA (Biosharp, BS043, China) in TBST buffer for 1 h and incubated overnight at 4 °C with the following primary antibodies: α-SMA (1: 500), COL-I (1: 500, AF0134, Affinity), TGF-β1 (1: 1000, AF1027, Affinity), proliferating cell nuclear antigen (PCNA) (1: 500, A12427, Abclonal, China), p-MEK1/2 (1: 500, AP0209, Abclonal), MEK1/2 (1: 500, A4868, Abclonal), p-ERK1/2 (1: 500, AF1015, Affinity), ERK1/2 (1: 500, AF6240, Affinity) and GAPDH (1: 10000, 60004-1-Ig, Proteintech, China). Next, the membrane was incubated with goat anti-mouse IgG (1: 3000, SE131, Solarbio) or goat anti-rabbit IgG (1: 3000, SE134, Solarbio) secondary antibody for 40 min at 37 °C. At last, the specific protein bands were visualized with Western ECL Substrate (D1010, Solarbio).

The lung tissues and ILC2s were lysed with radio immunoprecipitation assay lysis buffer (R0010, Solarbio, Beijing, China) on ice. Total protein was quantified via the BCA Protein Assay Kit (PC0020, Solarbio, Beijing, China). The protein was resolved using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; D1010, Solarbio, Beijing, China). The samples were transferred to polyvinylidene difluoride membranes (IPVH00010, Millipore, USA). Due to the stable positions of the target protein blots and in order to obtain the clearer bands, all the membranes were cropped prior to hybridization with primary antibodies. Then, they were blocked with 5% nonfat milk (A600669, Sangon, Shanghai, China). Subsequently, the membranes were incubated with anti-rabbit ST2 antibody (1:500; 11920-1-AP, ProteinTech, Wuhan, China) and anti-mouse GAPDH antibody (1:10000; 60004-1-Ig, ProteinTech, Wuhan, China) at 4°C. The next day, they were treated with the corresponding secondary antibody, HRP-labeled goat anti-rabbit IgG (1:3000; SE134, Solarbio, Beijing, China) or goat anti-mouse IgG (1:3000; SE131, Solarbio, Beijing, China) at 37°C for 1 h. The ST2 protein was visualized using ECL Western Blotting Substrate (PE0010, Solarbio, Beijing, China).

Total proteins from mouse skin tissues or HaCaT cells were prepared to separate using SDS/PAGE and then transferred onto PVDF membranes (IPVH00010; Millipore, Billerica, MA, U.S.A.). The membranes were incubated with primary antibodies overnight at 4°C, including p-JNK antibody (#4668; CST, Danvers, MA, U.S.A.), JNK antibody (#9252; CST), p-ERK antibody (#4370; CST), ERK (#4695; CST), p-IкB (#2859; CST), IкB (#9242; CST), p-p65 (#3033; CST), p65 antibody (#8242; CST), p-p38 antibody (bs-0636R; Bioss, Beijing, China), p38 antibody (bs-0637R; Bioss), and GAPDH antibody (60004-1-Ig; Proteintech, Wuhan, China). Then corresponding secondary HRP-conjugated goat anti-rabbit antibody (SE134; Solarbio) or goat anti-mouse antibody (SE131; Solarbio) was prepared to the membranes for 1 h at 37°C. After visualized using ECL reagent (PE0010; Solarbio), the immunoblots were imaged and the protein intensity was measured by Gel-Pro-Analyzer Software.

The proteins were extracted by using the RIPA lysate solution (Solarbio, Beijing, China); then, the equivalent amount of protein was separated by 5%, 8%, or 15% SDS-PAGE. After being transferred onto the PVDF membranes, the blots were blocked with fat-free milk for 1 hour. The membranes were then probed with primary antibodies such as CD63 antibody (1 : 1000 dilution, 25682-1-AP, Proteintech, Wuhan, China), CD81 antibody (1 : 2000 dilution, ab109201, Abcam, Cambridge, MA, USA), Cav3.2 antibody (1 : 500 dilution, 28358-1-AP, Proteintech), SERCA2 antibody (1 : 1000 dilution, DF6240, Affinity Biosciences, Zhenjiang, China), or GAPDH antibody (1 : 10000 dilution, 60004-1-Ig, Proteintech). The specific band was then detected by using a horseradish peroxidase- (HRP-) conjugated goat anti-rabbit antibody (1 : 3000 dilution, SE134, Solarbio) or goat anti-mouse-antibody (1 : 3000 dilution, SE131, Solarbio) followed by enhanced chemiluminescence (Solarbio).