Ba1055

HK-2 cells were lysed in radioimmunoprecipitation assay buffer (Beyotime, China). Immunoblotting was performed using the following rabbit primary antibodies: anti-CD9 (1:2,000; ab92726, Abcam, UK), anti-TSG101 (1:2,000; ab125011, Abcam), anti-HSP70 (1:1,000; ab2787, Abcam), anti-vimentin (1:1,000; ab92547, Abcam), anti-α-smooth muscle actin (anti-α-SMA; 1:1,000; ab7817, Abcam), anti-E-cadherin (anti-ECAD; 1:10,000; ab40772, Abcam), anti-cyclooxygenase 2 (anti-COX-2; 1:1,000; ab179800, Abcam), anti-AKT [1:1,000; 4691S, Cell Signaling Technology (CST), USA], anti-p-AKT (1:1,000; 4060S, CST), anti-mitogen-activated protein kinase (MAPK) (ERK1/2) (1:1,000; 8544S, CST), anti-p-MAPK (ERK1/2) (1:1,000; 8544S, CST), anti-p38 (1:1,000; 8690S, CST), anti-p-p38 (1:1,000; 9216S, CST), anti-SOX2 (1:1,000; AF5140, Affinity, Canada), and anti-β-actin (1:5,000; ab8226, Abcam). Horseradish-peroxidase-conjugated goat anti-rabbit immunoglobulin G (IgG) (1:5,000; BA1055, Boster, China) and horseradish-peroxidase-conjugated goat anti-rat IgG (1:5,000; S0009, Affinity, Canada) were used as the secondary antibodies. A chemiluminescence imaging system (ChemiScope 6200T, Clinx Science Instruments, Shanghai, China) was used for detection.

Cells were cultured in 24-well plates and lysed with NP40 lysis buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, and 0.5% NP-40) supplemented with a protease inhibitor cocktail and PMSF (1 mM) for 30 min at 4°C. The cell lysates were centrifuged for 10 min at 12,000 g and 4°C. The supernatants were transferred into a new tube, resolved by sodium dodecyl sulfate gel electrophoresis (SDS-PAGE), and transferred to PVDF membranes (Bio-Rad) to measure protein expression. The membrane was blocked with TBS-T buffer (10  mM Tris, pH 7.4, 150  mM NaCl, 0.1% Tween-20) containing 5% nonfat dry milk for 1  h at room temperature. Primary Antibody Dilution Buffer (Beyotime, P0023A-100ml) is used to dilute primary antibodies to working concentrations, then the membranes were incubated with primary antibodies at 4°C overnight. The HRP-conjugated goat antiMouse IgG secondary antibodies (Boster, Wuhan, China, BA1051) or HRP-conjugated goat antiRabbit IgG secondary antibodies (Boster, BA1055) were diluted in TBS-T (1:5,000 ratio). Next, the membranes were incubated with secondary antibodies for 1 h at room temperature. The blots were developed using the BeyoECL Plus (Beyotime, P0018S) and imaged with an Amersham Imager 600 (GE Healthcare) imaging system.

Western blotting and cell viability measurements were performed as previously specified.¹², ¹³ Anti‐Nucleolin rabbit antibody was obtained from Sigma (1:1000, N2661), anti‐Aurora B rabbit antibody from Cell Signaling Technology (1:1000, 3094T) and anti‐β‐Actin mouse antibody also from Sigma (1:1000, A2228). Anti‐rabbit secondary antibody (1:10 000 dilution, BA1055, Boster) to detect nucleolin protein and aurora B protein and anti‐mouse secondary antibody (1:2500 dilution, BA1050, Boster) to detect β‐Actin protein.

Skin tissue was homogenized in RIPA (Radio Immuno Precipitation Assay) lysis buffer, protease inhibitor, and phosphatase inhibitor (Boster, Wuhan, China) to extract the total protein. The protein concentration was determined using BCA protein assay kit. The protein was denatured in gel sample buffer at 100 °C for 5 min, separated by 10% SDS-PAGE, transferred to a polyvinylidene fluoride (PVDF) membrane (EpiZyme, Shanghai, China), and blocked with 5% (w/v) non-fat dry milk in TBST buffer (Tris-Buffered Saline with Tween-20). The membrane was incubated with the primary antibody against NF-κВ (BA1297-2, Boster, Wuhan, China), TNF-α (BA14901, Boster), IL-6 (BA4339-2, Boster), COX-2 (BA3708, Boster), ERK (GB112238, Servicebio, Wuhan, China), JNK (M02608-3, Boster), P38 (A00176-2, Boster), AP-1 (GB11270, Servicebio), MMP-1 (A00733-1, Boster), and MMP-2 (A00286, Boster) at 4 °C overnight, followed by washing at TBST, and then incubated with goat anti-rabbit HRP (horseradish peroxidase)-IgG (BA1055, Boster) as the secondary antibody for 45 min. After washed with TBST 3 times for 10 min, membrane was incubated in SuperSignal ECL Western substrate (Biosharp, Hefei, China). The protein band was visualized using the Chemiluminescent Imaging System (Tanon-5200SF, Shanghai, China). The intensity was quantified by Image J version 1.8.0 program.

Proteins were isolated using RIPA buffer (P0013B, Beyotime) with protease inhibitor (ST505, Beyotime) and phosphatase inhibitor (P1081, Beyotime), and the concentrations were measured using a bicinchoninic acid (BCA) kit (P0012S, Beyotime). Lysates (60 μg/lane) were separated by 12% SDS-PAGE gels and subsequently transferred to PVDF membranes. The membranes were blocked with 5% BSA for 1 hour at room temperature and subsequently incubated with corresponding antibodies as follows: GAPDH (1 : 1000, ab8245, Abcam), T-AKT (1 : 1000, 2920, CST), p-AKT (1 : 1000, 4060, CST), PTEN (1 : 1000, 9188, CST), MMP-9 (1 : 1000, 3852, CST), Vimentin (1 : 1000, 3932, CST), caspase 3 (1 : 1000, 14220, CST), cleaved caspase 3 (1 : 1000, 9664, CST), Bcl-2 (1 : 1000, 60178, Proteintech), or Bax (1 : 1000, 60267, Proteintech) at 4°C overnight. After incubation, the membranes were washed three times with 0.1% TBST and incubated with HRP-conjugated goat anti-mouse (BA1051, Boster) or goat anti-rabbit (BA1055, Boster) secondary antibody for 1 hour at room temperature; the signal was detected using enhanced chemiluminescence reagent (P0018S, Beyotime). The intensity of each band was measured using the Imaging J System (Bio-Rad, USA).

Lung tissue and cellular protein were extracted using RIPA buffer (EpiZyme, PC101, Shanghai, China) with 0.1% PMSF, lysed on ice for 30 min, and centrifuged at 12,000 r for 10 min. The protein was quantified using a BCA Kit, and a 30 μg protein sample was obtained for 12-15% SDS-PAGE electrophoresis and transferred onto the PVDF membrane and then probed with rabbit anti-ENO2 (Bioss, bs-6273R, Beijing, China), Rabbit anti-SP (Beyotime, AF8094, Shanghai, China), Rabbit anti-VIP (Beyotime, AF8331, Shanghai, China), or Rabbit anti-CGRP (Beyotime, AF6495, Shanghai, China) primary antibodies overnight at 4×C. The secondary antibody of horseradish peroxidase (HRP)-goat anti-rabbit immunoglobulin G (IgG) (Boster, BA1055, Wuhan, China) was diluted to 1 : 3,000. The signals were detected using ECL (Millipore, WBLUR0100, Millipore, USA). The bands were visualized with the chemiluminescence detection system (Tanon 5200 Multi, Beijing, China), and the protein expression levels were normalized to GAPDH levels. The integrated density of proteins was quantified using ImageJ software (NIH).