Matrix metallopeptidase 9 mmp 9

EA, TBA, DTNB, HEPES were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Antibodies against BCL2, Bax, p53, p21, Matrix metallopeptidase-9 (MMP-9), and anti-rabbit IgG fluorescein isothiocyanate (FITC) and Rhodamine 1,2,3 conjugated secondary antibodies, and 4′,6-diamidino-2-phenylindole (DAPI) were obtained from Santa Cruz (Santa Cruz, CA, USA). 2′,7′-Dichlorofluoroscein diacetate (H₂DCF-DA) was purchased from Calbiochem of Merck-Millipore (Billerica, MA, United States). Antibody against p-NF-κB, p-STAT3, cyclooxygenase-2 (COX-2) and p-Akt were purchased from Cell Signaling (Beverly, MA, USA). Antibody to detect the level of active caspase 3, annexin-FITC-PI, and JC1 dye were purchased from Becton Dickinson-Biosciences (San Jose, CA, USA). All the cell culture reagents were purchased from Gibco (Waltham, MA, US) and all other reagents used for this study were of highest quality grade.

Total extracted protein lysates from similar portions of LV tissue were prepared by standard procedures, and protein lysate concentrations were determined via BCA protein assay kit (Beyotime, Shanghai, China). Equivalent amounts of tissue protein (50 μg) were subjected to 10–12% SDS-PAGE, electrotransferred onto polyvinylidene difluoride (PVDF) membranes, and then incubated overnight at 4°C with the primary antibodies, including transforming growth factor-β1 (TGF-β1), transforming growth factor-β receptor II (TGFβRII), p-Smad2, p-Smad3, Smad2, Smad3, Smad4 (Cell Signaling Technology, USA), matrix metallopeptidase-2 (MMP-2), matrix metallopeptidase-9 (MMP-9), tissue inhibitor of metalloproteinases-2 (TIMP-2), and GAPDH (Santa Cruz biotechnology, USA). Next day, membranes were washed and incubated with the corresponding secondary antibodies for 2 h; anti-mouse and anti-rabbit antibodies were from Cell Signaling Technology; protein bands were detected by Fluor Chem E (Protein Simple, USA). Quantitation was performed via Image J software (Bethesda, MD, USA). GAPDH was used as loading controls for total protein expression.

Elacridar and dithiothreitol (DTT) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco’s modified Eagle’s medium (DMEM), sterile phosphate-buffered saline (PBS), and penicillin/streptomycin antibiotics were obtained from Gibco (Grand Island, NY, USA). Fetal bovine serum (FBS) was purchased from Atlanta Biologicals (Flowery Branch, GA, USA). Total protein measurement reagents using the bicinchoninic acid (BCA) method were obtained from Pierce (Rockford, IL, USA). Antibodies used were mouse monoclonal antibody against light chain LRP1 (Abcam, Cambridge, MA, USA), mouse monoclonal antibody C-219 against P-gp from BioLegend (San Diego, CA, USA), mouse monoclonal antibody against BCRP (Cell signaling; Boston, MA, USA). Monoclonal antibodies for claudin-5, ZO-1, and HRP-labeled secondary antibodies were purchased from Invitrogen (Carlsbad, CA, USA), goat polyclonal antibodies against actin (C-11) and Matrix Metallopeptidase 9 (MMP9) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Antibodies for pre- and postsynaptic proteins SNAP-25 and PSD-95, respectively, were purchased from GeneTex (Irvine, CA, USA), antibodies for IκB-α, p-IκB-α, NF-кB p65, and p-NF-кB p65 were purchased from Cell Signaling. All other reagents and supplies were purchased from VWR (West Chester, PA, USA) and Fisher Scientific (Hampton, NH, USA).