Goat anti rabbit igg h l hrp conjugated antibody

Sodium butyrate, propionate, TSA, WST-1, and a protease inhibitor cocktail were purchased from Sigma-Aldrich. Butyrate and propionate were dissolved in endothelial growth factor (EGM-2) and TSA in DMSO and then further diluted in EGM-2. Concentrations used were based on recent publications of our group [5 (link),6 (link)]. Human recombinant TNFα was bought from eBioscience. The human IL-8 enzyme-linked immunosorbent assay (ELISA) kit, Lipofectamine 2000 (lipo-2000), and the BLOCK-iT Alexa Fluor Red Fluorescent control were purchased from Invitrogen. The NF-κB p65 (pS536) ELISA kit was bought from Cell signaling technology. The IL-33 ELISA kit was purchased from U-CyTech biosciences. The IL-33 monoclonal antibody and Sliencer GAPDH siRNA (human) were bought from Thermo Fisher Scientific. Silencer pre-designed on-Targetplus SMARTpool siRNA IL-33 and siGENOME Non-Targeting siRNA pool (Silencer negative control) were bought from Dharmacon. The following monoclonal antibodies: the anti-phospho p38 (phospho T180 + Y182) antibody, the anti-JNK1+JNK2 (phospho T183 + Y185) antibody, the anti-ERK1/2 (phospho Thr202/Tyr204) antibody, the anti-GAPDH antibody, the rabbit anti-mouse IgG H&L (HRP) conjugated antibody, and the goat anti-rabbit IgG H&L (HRP) conjugated antibody were purchased from Cell Signaling Technology and Abcam.

4T1 cells were seeded in 6-well plates and incubated overnight at 37 ℃. Different drug formulations were then added. After 24 h incubation, the cells were washed twice with PBS and incubated with RIPA lysis buffer for 10 min. The supernatant was collected after centrifugation (12,000 rpm) at 4 ℃ and the protein concentration was measured by BCA Assay Kit. After separation by gel (10% SDS-PAGE) electrophoresis, the proteins were electrotransferred to PVDF membranes, blocked with 5% skim milk for 1 h, and incubated with antibodies against β-tubulin (1:1000, Cell Signaling), MRP2 (1:1000, Cell Signaling), HIF-1α (1:1000, Cell Signaling), MMP9 (1:1000, Cell Signaling) and RKIP (1:1000, Cell Signaling) overnight at 4 ℃. PVDF membranes were then washed with TBST for 3 times and incubated with goat anti-rabbit IgG (H + L), HRP conjugated antibody (1:2000, Cell Signaling) for 1 h at room temperature. Finally, the expression of protein was detected by GeneGenome5 chemiluminescence system (Syngene, Cambridge, UK).

Minimal Essential Medium (MEM) and Dulbecco’s Modified Eagle Medium/F12 (DMEM/F12) with glutamax were purchased from Gibco (Thermo Fisher Scientific, Breda, Noord-Brabant, The Netherlands). Fetal bovine serum (FBS) was purchased from Bodinco (Alkmaar, Noord-Holland, The Netherlands). The bovine collagen type I solution was obtained from Advanced BioMatrix (CellSystems Biotechnologie Vertrieb, Troisdorf, Germany). The sodium salts of propionate and butyrate, as well as fibronectin from human plasma and penicillin–streptomycin (pen-strep) solutions, were purchased from Sigma (Breda, Noord-Brabant, The Netherlands). Sodium acetate was obtained from Merck Millipore (Amsterdam-Zuidoost, Noord-Holland, The Netherlands). Recombinant human IL-13 were purchased from R&D Systems (Abingdon, Oxfordshire, UK). Recombinant human IL-4 was purchased from ProSpec-Tany TechnoGene (Ness-Ziona, Israel). The HDM extract was obtained from GREER (Lenoir, NC, USA). The following monoclonal antibodies: anti-JNK1 + JNK2 (phospho T183 + Y185) antibody, anti-ERK1/2 (phospho Thr202/Tyr204) antibody, anti-GAPDH antibody, rabbit anti-mouse IgG H&L (HRP) conjugated antibody and goat anti-rabbit IgG H&L (HRP) conjugated antibody were purchased from Cell Signalling Technology (Leiden, Zuid-Holland, The Netherlands) and Abcam (Cambridge, UK).