Alliance q9

Proteome profiler rat cytokine array kit, panel A (R&D Systems, Minneapolis, MN, catalogue no. ARY008) was used to evaluate the difference in cytokine levels in each group of rats. Each spot of the membrane was coated with 29 types of specific antibodies of rat cytokine, including CXCL1/CINC-1, IL-1ra/IL-1F3, l-selectin, CXCL3/CINC-2α/β, IL-2, CXCL9/MIG, CXCL2/CINC-3, IL-3, CCL3/MIP-1α, CNTF, IL-4, CCL20/MIP-3α, fractalkine/CXC3CL1, IL-6, CCL5/RANTES, GM-CSF, IL-10, CXCL7/thymus chemokine, ICAM-1, IL-13, TIMP-1, IFN-γ, IL-17, TNF-α, IL-1α/IL-1F1, CXCL10/IP-10, VEGF, IL-1β/IL-1F2 and LIX. According to the manufacturer’s protocol, a pooled plasma sample from five rats in each group was mixed with a detection antibody cocktail and incubated for 1 h at room temperature. During this period, the membrane was incubated with a blocking buffer for 1 h at room temperature and then soaked with a pre-mixed pooled plasma sample overnight at 4 °C. The membranes were then subsequently incubated with HRP-conjugated streptavidin for 30 min at room temperature. The signal was evaluated after exposure to peroxidase substrate using Alliance Q9 (Uvitec, Cambridge, UK). The intensity of the signal was scored using UVITEC Alliance software (Uvitec, Cambridge, UK). The data were expressed as the fold change with correspondence to the normal group.

Under stereomicroscope, substantia nigra and striatum were isolated and the different regions were freshly lysate using pestles, protein extracted were dosed as previously described [43 (link)]. Tissues lysates containing 10μg of protein were separated on 4–13% gradient Bis-Tris gel in running buffer at 100 mV for 80 min. Proteins were transferred into PVDF membranes using a semi-dry device (Thermo scientific, UK). Membranes were washed in tris-buffered saline with 0.05% Tween20, and blocked in 5% no-fat milk for 1 h at RT. Membranes were then incubated overnight at 4°C with the following primary antibodies, diluted in the same blocking solution: anti p-NRF2 (1:5000 Abcam, UK), anti-NFKB (1:2000 Abcam, UK) anti-p-TRKB (1:2000 Cell Signaling), anti-BDNF (1:500 Abcam, UK), anti-PPARγ (1:500, Thermo, USA) anti-HO1(1:1000 Santa Cruz, USA) at 4°C overnight and then incubated with 1:10000 HRP-conjugated anti-rabbit IgG or anti-mouse IgG. Protein bands were detected with West Pico luminol (Thermo scientific) following kit’s datasheet. Through Alliance Q9 (Uvitec, Cambridge, UK) image chemiluminescent bands were detected and using ImageJ program we analyzed each band intensity normalized as indicated in the “Wester Blotting” in vitro section.

OxyBlot kit (Merck Millipore, Burlington, MA, USA) was performed according to the manufacturer’s instructions to evaluate oxidized protein levels. In brief, cells were plated (density 1.5 × 10⁴ cells/cm²) in 10% FBS-supplemented medium. 24 h after seeding, the cells were exposed to our differentiation conditions, as previously mentioned. After complete differentiation, cells were exposed for 24 h to co-treatment with GSK0660 0.2µM, and 25µM 6-OHDA in culture media supplemented with FBS 1%, as previously described. Consequently, pellets were collected and homogenized in lysis buffer having dithiothreitol (DTT) 50mM. 5 µg/µl of protein extracts were derivatized following manufacturer’s protocols. 20 µg total proteins per sample were separated on 10% polyacrylamide SDS denaturing gels. As primary antibody rabbit anti-DNP diluted 1:150 was incubated. According to manufacturer’s instructions, after incubation with secondary HRP-conjugated anti-rabbit IgG antibody diluted 1:300, the immunoreactive bands were detected with West Pico luminol (Thermo Scientific). Bands were visualized using Alliance Q9 (Uvitec, UK) and analyzed by FIJI software. As housekeeping protein actin was used, and values were given as relative units (R.U.).

Under stereomicroscope, substantia nigra and striatum were isolated and lysate using pestles to extract and quantify proteins as reported by [30 ]. 20 µg of proteins were run on 4–13% Nupage Bis-Tris precast gel (Thermo Scientific, USA) in running buffer at 200 V for 1 h, then transferred onto PVDF membranes using a semi-dry device (Thermo Scientific, USA) for 10 min in 1 Step buffer. Membranes were incubated with Blocking Buffer (Thermo Scientific, USA) for 15 min at RT and then incubated O/N at 4 °C with the following primary antibodies: anti-TH (1:1000), anti-pCreb (Ser133) (1:500), anti pAKT (Ser473) (1:1000) and anti-mBNDF (1:1000), diluted in the same blocking solution: at 4 °C O/N and then incubated with 1:10000 HRP-conjugated secondary antibody. Protein bands were detected with West Pico luminol (Thermo scientific) using Alliance Q9 (Uvitec, UK). For the densitometric analyses, ImageJ was used and normalized upon the housekeeping protein (HRP-conjugated actin).

For siRNA experiments, cells were plated in 96-well plates at 6000 cells per well and treated with compounds 24 h later. The cells were transfected with 15 nM siRNA against KLF5 (ONTARGETplus SMART-POOL®, Dharmacon) or with a control siRNA at the respective concentration (ONTARGETplus SMART-POOL®, Dharmacon) using Lipofectamine® RNAiMAX (Invitrogen) following the manufacturer’s protocol. Alternatively, for siRNA experiments against NEK3 and TTC7A, and for additional KLF5 experiments, cells were plated in 6-well plates at 400,000 cells per well and treated with compounds 24 h later. The cells were transfected with 30 nM against NEK3 and TTC7A or with 5 nM and 10 nM against KLF5; 48 h post seeding, the cells were split and plated in 96-wells at 10,000 cells per well. The effect of the knockdown against KLF5, NEK3, or TTC7A on cell viability/proliferation was measured by live cell imaging using Incucyte® (Satorius) or by the MTT assay (Sigma M2128) at 72 h (or at the indicated time point) post-transfection; 500 µg/mL MTT salt was diluted in complete medium and incubated at 37°C for 2 h. Formazan crystals were extracted using 10% Triton X-100 and 0.1 N HCl in isopropanol, and color absorption was quantified at 570 nm and 630 nm (Alliance Q9, Uvitec).

Conditioned media from two PRODH-expressing and two control clones from the NCI-H1299 cell line were analyzed with the C-series Human Cytokine Antibody Array C100 (Human Cytokine Array C6 and Human Cytokine Array C7, RayBiotech, Inc., Norcross, GA, USA).
Secretome profiling was performed using 50 µg of total protein from the conditioned media after processing as described above. After incubation, the chemiluminescent signals developed by HRP were detected using an Alliance Q9 (Uvitec) instrument. Signals were quantified by ImageJ software, version 1.53q, National Institute of Health. Two biological replicates were performed for each clone.