Phospho p38

A standard Western blotting protocol was used as described previously [5 (link),25 (link)] with antibodies against TGFβ (1:600), matrix metalloproteinase 2 (MMP2, 1:200) (Santa Cruz, CA, USA); connective tissue growth factor (CTGF, 1:500) (Biovision, Mountain View, CA, USA), LOX-1 (1:1000) (R&D System), bone morphogenetic protein-7 (BMP-7, 1:5000) (AbDSerotec, Minneapolis, MN, USA), nuclear factor-kappa B (NFκB, 1:1000), its inhibitor kappa B alpha (IκBα, 1:200), Phospho-P38 (1:1000) (Millipore, Billerica, MA, USA), NADP(H) oxidase subunit Gp91phox (1:500, BD Transduction Laboratories, France). Loading controls were β actin (1:3000, Sigma Aldrich, France) or P38 (1:1000, Millipore). Appropriate HRP-coupled secondary antibodies (1:5000 to 1:10 000, GE Healthcare, France) were used to detect the band by chemiluminescence with ECL plus (GE Healthcare, France). Intensities of the protein bands were determined and quantified using AlphaEase FC software (Alpha Innotech Corporation, San Leandro, CA).

Luteolin (≥98% purity by TLC), cell counting kit-8 assays (CCK-8), and DAPI solution were purchased from Sigma-Aldrich (St. Louis, MO, USA). Human recombinant proinflammatory cytokine IL-1β and enzyme-linked immunosorbent assay (ELISA) kits were purchased from R&D Systems (Minneapolis, MN, USA). The inhibitors PD98059, SP600125, SB202190, and Bay 117082 were purchased from Enzo Life Sciences (Farmingdale, NY, USA). Antibodies against β-actin, COX-2, iNOS, HO-1, AKT, and phosphorylated- (phospho-) AKT were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against JNK, ERK, p38, phospho-JNK, phospho-ERK, and phospho-p38 were purchased from Millipore (Billerica, MA, USA).

To analyze the signaling transduction pathways, western immunoblotting was performed as previously described [42 (link)]. Briefly, the BMMCs were treated with different mTORi (rapamycin: 10 nM; everolimus: 10 nM; temsirolimus: 2 µM) or DMSO (control group) to generate immature BMM-derived DCs and then co-cultured with TC-1 tumor cells on day 6 for 6 h. All groups were lysed in PhosphoSafe™ Extraction Reagent (Novagen, Merck KGaA, Darmstadt, Germany) with a proteinase inhibitor (Sigma, Merck KGaA, Darmstadt, Germany). The protein extracts were quantified with a BCA Protein Assay Kit (Pierce, Rockford, IL, USA).
These targeting proteins were separated by SDS/PAGE (10% gel), transferred onto an PVDF membrane (Millipore, Merck KGaA), and probed with primary antibodies specific to mTOR (Genetex, Irvine, CA, USA), phospho-mTOR (Genetex), Bad (Genetex), Bak (Genetex), p38 (BioLegend), phospho-p38 (Millipore) and GAPDH (Abcam, Cambridge, MA, USA). Then, HRP-conjugated second antibodies (Hycult Biotech, Uden, The Netherlands) were probed and the specific bands were visualized by ECL^® Western blotting system (GE Healthcare, Salt lake city, UT, USA). The band density was quantified with ImageJ and the relative ratios of the indicated proteins/GAPDH were shown.