Sc 94

Anti-phospho-ERK (1:500, sc-7383, Santa Cruz Biotechnology), anti-ERK (1:500, sc-94, Santa Cruz Biotechnology), anti-phospho-p38 (1:1000, 9211, Cell Signaling Technology), anti-p38 (1:1000, 9212, Cell Signaling Technology), anti-HMGB1 (1:1000, ab18256, Abcam), anti-beta-actin (1:5000, ab6276, Abcam), and anti-VEGF (1:1000, ab46154, Abcam) were used as primary antibodies. Cells were lysed in RIPA buffer containing protease and phosphatase inhibitor on ice for 45 min and collected by centrifugation at 16000×g for 15 min at 4 °C. Protein concentrations were measured by the bicinchoninic acid protein assay kit (Pierce). Cell lysates containing 30 µg of total protein were separated by 10% SDS-PAGE and subsequently transferred to nitrocellulose membranes. The membranes were subsequently probed with the indicated antibodies and proteins were detected using the ECL Plus Western Blotting Detection System (GE Healthcare).

ECFCs were lysed in radioimmunoprecipitation assay (RIPA) buffer in the presence of protease (Complete Ultra, Roche, Penzberg, Germany) and protein phosphatase inhibitors (sc-45045 and sc-45065, Santa Cruz Biotechnology, Santa Cruz, US). Proteins were separated by SDS-PAGE and immunolabelled using specific antibodies for ERK1/2 (sc-94, Santa Cruz Biotechnology) and phospho-ERK1/2 (9101S, Cell Signaling). Extracts from cell-free hPLG-coated wells were utilized as negative controls, which highlighted the presence of dephosphorylated ERK1/2 from platelets. This did not interfere with the analysis of ERK1/2 phosphorylation. Blots were stained with IRDye 800CW or IRDye 680RD secondary antibodies and scanned with a LI-COR Odyssey CLx infrared imaging system. Densitometric analyses were performed using Image Studio Lite v5.0.21 (LI-COR, Lincoln, US).

PA was purchased from ChemFaces (Wuhan, China). A stock solution of Paeruptorin A (PA) was made at a concentration of 100 mM in dimethyl sulfoxide (DMSO) and stored at −20 °C. Antibodies against cyclin D1 (sc-717), p21 (sc-397), Skp2 (sc-7164), p27 (sc-528), TIMP-1 (sc-5538), TIMP-2 (sc-5539), p-ERK1/2 (sc-16982), ERK (sc-94), p-JNK (sc-6254), JNK (sc-571), p-p38 (sc-17852-R), p38 (sc-7972) and β-actin (sc-47778) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). MMP-2 (ab92536) and MMP-9 (ab137867), HRP-conjugated anti-mouse (sc-516102) and anti-rabbit (sc-2004) secondary antibodies were purchased from Abcam (Cambridge, UK). The MEK1/2 inhibitor PD98059 was purchased from Calbiochem (San Diego, CA, USA). MTT was purchased from Sigma (St. Louis, MO, USA). All stock solutions were wrapped in foil and stored at −20 °C until use.

Duodenal samples were lysed on ice in buffer containing 10 mM HEPES [pH 7.9], 10 mM KCl, 0.1 mM EthyleneDiamineTetraacetic Acid (EDTA), 0.2 mM Ethylene Glycol-bis (β-aminoethyl ether)-N,N,N',N'-Tetraacetic Acid (EGTA), and 0.5% Nonidet P40 supplemented with 1 mM dithiothreitol (DTT), 10 mg/ml aprotinin, 10 mg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM Na₃VO₄, and 1 mM NaF. Lysates were clarified by centrifugation and separated on sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis. Blots were incubated with antibodies against SMAD7 (1 μg/ml MAB2029, R&D Systems, Minneapolis, MN, USA), total ERK (sc-94, Santa Cruz Biotechnology, Santa Cruz, CA, USA), p-SMAD2,3 (#8828, Cell Signaling Technology, Danvers, MA, USA), total and phosphorylated STAT4 proteins (sc-486, sc-28296, Santa Cruz Biotechnology). Densitometry data for SMAD 7 and SMAD2/3 was normalized using ERK1/2 by calculating values expressed as arbitrary units (a.u.) using ERK1/2 as the denominator. Multiple Western blots were performed (blots were stripped and then incubated with different antibodies), and densitometry data from each Western blot were taken individually for analysis given inherent variability in densitometry as is established [24 (link)]. Limited availability of tissue samples made it impractical to perform transcriptional-level analyses using RNA.