Erk 16443 1 ap

Recombinant human PN-1 was purchased from R&D Systems (2980-PI-010). Recombinant human IL-1β (AF-200-01B-10), TNF-α (AF-300-01A-10), and TGFβ1 (AF-100-21C-10) were obtained from Peprotech. Polyclonal antibodies to PN-1 (ab154591), MMP13 (ab39012), ADAMTS5 (ab135656), and Aggrecan (ab3778) were purchased from Abcam. Mouse anti-human fibronectin N-terminal monoclonal antibody (mABl936) was obtained from Chemicon (San Francisco, CA, USA). Polyclonal antibodies against COL2 (sc-7764) and P-P65 (sc-33020) were obtained from Santa Cruz. Polyclonal antibodies against P38 (BS1681), P-P38 (BS6381), P-ERK (BS5016), and MMP9(BS6893) were obtained from Bioworld. P65 (10745-1-AP), and ERK (16443-1-AP) were purchased from ProteinTech Group. The anti-MMP3 (14351) antibody was purchased from CST and the anti-ADAMTS4 (EAP1002) antibody was obtained from Elabscience. All fluorescence-conjugated secondary antibodies, anti-IgG horseradish peroxidase (HRP)-conjugated antibodies, and the LaminB antibody were obtained from BosterBio. DAPI was purchased from Beyotime Biotechnology.

Following lysis of the cells in the presence of protease and phosphatase inhibitors (Roche Diagnostics, Indianapolis, IN, USA), whole-cell lysates underwent SDS-polyacrylamide gel electrophoresis, with electrophoretic transfer onto polyvinylidene difluoride membranes (Thermo Fisher Scientific, Rockford, IL, USA), and immunoblotted using anti-Mcl-1 (16225-1-AP), -Bcl-2 (12789-1-AP), -Bcl-xL (66020-1-Ig), -Bax (50599-2-Ig), -β-actin (66009-1-Ig), -ERK (16443-1-AP) (Proteintech, Rosemont, IL, USA), -p-AKT (T308; AF0832), -p-AKT (S473; AF0016) (Affinity Biosciences, Zhenjiang, Jiangsu, China), -Bim (2819), -cf-Caspase 3 (9661), -p-STAT5(Y694; 9359 S) (Cell Signaling Technologies, Danvers, MA, USA), -Bak (ab69404), -p-ERK(T202/Y204; ab4819), -AKT (ab8805), -FLT3 (ab245116) (Abcam, Cambridge, MA, USA), as previously described^{22 (link),23 (link)}. The Odyssey Infrared Imaging System (Li-Cor, Lincoln, NE, USA) was used to visualize immunoreactive proteins, as described by the manufacturer. Western blots were repeated, at a minimum three times, and one representative blot is displayed. The Odyssey V3.0 program (Li-Cor) was used to perform densitometry measurements, normalized to β-actin, and calculated as fold-change compared to the corresponding vehicle control.

RIPA Buffer (Tris–HCl 25 mM pH 7.4, NaCl 150 mM, 1% Triton X-100, Sodium Deoxycholate 1%, SDS 0.1%, EDTA 2 mM) containing a protease/phosphatase inhibitor mixture (Roche) was utlized to lyse cells. After electrophoresis with SDS-PAGE, the separated proteins from the gel (50 μg) were transferred on nitrocellulose (NC) membranes, followed by incubation with primary antibodies. Following incubation with appropriate HRP-labeled secondary antibodies, ECL HRP substrate (Beyotime) was employed to detect signals. The primary antibodies used were as follow: α-SMA (55135-1-AP, Proteintech), Collagen I (14695-1-AP, Proteintech), Timp-1 (CSB-PA023560YA01HU, CUSABIO, Wuhan, China), Vimentin (10366-1-AP, Proteintech), Erk (16443-1-AP, Proteintech), p-Erk (sc-81492, Santa Cruz Biotechnology, Santa Cruz, CA, USA), p38 (14064-1-AP, Proteintech), p-p38 (AF4001, Affinity, Changzhou, China), JNK (AF6319, Affinity), p-JNK (AF3318, Affinity), NF-κB (AF5006, Affinity), p-NF-κB (AF2006, Affinity).