Mmp 1 elisa duoset

Recombinant human RANTES/CCL5, Met-RANTES and IL-1β, and MMP-1 ELISA Duoset were purchased from R&D systems (Minneapolis, MN, USA). MMP-13 ELISA was purchased from Ray Biotech (Norcross, GA, USA). MMP-1 and MMP-13 SYBR Green primers were purchased from Qaigen (Valencia, CA, USA). SMARTpool ON-TARGETplus RANTES small-interfering RNA (siRNA) and On-target plus non targeting siRNA (D-001810-10) control were purchased from GE Dharmacon (Lafayette, CO, USA). MMP-1, MMP-13, type I collagen, and lamin A/C antibodies were purchased from Santa Cruz Biotech (Santa Cruz, CA, USA). Rabbit monoclonal or polyclonal antibodies against p-p38 (Thr¹⁸⁰/Tyr¹⁸²), p-ERK (Thr²⁰²/Tyr²⁰⁴), p-JNK(Thr¹⁸³/Tyr¹⁸⁵), p-PKCδ (Thr⁵⁰⁵), total p-38, total JNK, total ERK, p-c-Jun (Ser⁷³), and p-ATF-2 (Thr^69/71) were from Cell Signaling Technologies (Beverly, MA, USA). Rabbit polyclonal antibodies against CCR1 and CCR5 were from BioVision (Milpitas, CA) and GeneTex (Irvine, CA, USA), respectively. Heparinase III (flavobacterium heparinum derived), heparan sulfate degrading lyase was purchased from Sigma (St. Louis, MO, USA). type I collagen was purchased from Advanced Biometrix (Carlsbad, CA, USA). Inhibitors of JNK (SP600125), ERK (PD98059), p38 (SB 02190), PKCδ (Rottlerin), and NF-κB (PDTC) were purchased from EMD Millipore (Billerica, MA, USA).

NHDF fibroblasts were seeded in 96-well plates (4500 cells/well) in DMEM medium supplemented with 3% FCS. The medium was changed to one containing the different dietary compounds at the indicated concentrations for one day. TNFα (1.5 ng/mL) was then added, and the medium was collected after 24 h. The amount of MMP-1 protein in the medium was measured using a human total MMP-1 ELISA DuoSet (R&D systems, Minneapolis, MN, USA).