Anti pstat3 tyr705

Tissue specimens and cells were lysed in RIPA buffer (Beyotime, China) and the protein concentrations were measured by a BCA kit (Beyotime, China). Equal amounts of proteins were separated on 10% SDS-PAGE and transferred to polyvinylidene fluoride membranes. After washing and blocking with sealing fluid, all membranes were incubated with rabbit polyclonal anti-RIOK1 (1: 2,000 dilution, Abcam, Cambridge, UK), anti-Cyclin B1 (1:1,000 dilution, Cell Signaling Technology, #4135), anti-p-AKT Ser473 (1:1,000 dilution, Cell Signaling Technology, #4060), anti-AKT (1:1,000 dilution, Cell Signaling Technology, #4691), anti-MMP2 (1:1,000 dilution, Cell Signaling Technology, #40994), anti-N-cadherin (1:1,000 dilution, Cell Signaling Technology, #13116), anti-vimentin (1:1,000 dilution, Cell Signaling Technology, #5741), anti-Cleaved PARP (1:1,000 dilution, Cell Signaling Technology, #5625), anti-Cleaved Caspase-3 (1:1,000 dilution, Cell Signaling Technology, #9664), anti-stat3 (1:1,000 dilution, Cell Signaling Technology, #12640), anti-p-stat3 Tyr705 (1:1,000 dilution, Cell Signaling Technology, #9145), anti-twist (1:1,000 dilution, Cell Signaling Technology, #69366), anti-E-cadherin (1:1,000 dilution, Abcam, Cambridge, UK), Tubulin (1:1,000 dilution, Abcam, Cambridge, UK). And then the corresponding secondary antibodies were incubated. ECL kit was used for detection.

The formalin-fixed, paraffin-embedded tissue was sectioned (4 μm) and mounted onto poly-L-lysine-coated glass slides for immunohistochemistry [13 (link)]. After being deparaffinized in xylene and rehydrated in a series of graded ethanol solutions, the slices were heated in a high-pressure cooker with 10 mmol/L of citrate buffer (pH 6.0) for antigen retrieval. The anti-pSTAT3^Tyr705 (1 : 500, #9145; Cell Signaling, Danvers, MA, USA) was incubated with tissue sections at 4°C overnight. Subsequently, the slices were incubated with a secondary antibody and color-developed using DAB according to the manufacturer's recommendations.

We assessed the A009 extract ability to target STAT3 phosphorylation, as a biochemical consequence of their cancer preventive and angiopreventive properties. Following 24 h of treatment with A009 extract (1:250, batch 4 or 3) or HyT alone at the same concentration present in A009 dilutions, the cells were lysed in RIPA buffer, supplemented with protease and phosphatase inhibitor cocktails (Roche Diagnostics GmbH). Proteins (50 μg) were separated on the NupageNovex on 4–12% Bis-Tris Gel (Life Technologies) and transferred to a PVDF membrane Amersham Hybond (GE Healthcare Biosciences). Membranes were incubated overnight at 4 °C with anti-p-STAT3 (Tyr705) (Cell Signaling Technology) and with peroxidase-linked anti-rabbit IgG secondary antibodies (GE Healthcare Life science) for 1 h at room temperature. Specific protein bands were detected with Pierce ECL Western Blotting Substrate (ThermoFisher Scientific). Band intensity (revealed as optical density-OD) was quantified using ImageJ software, v 1.52. Every band was normalized versus the respective housekeeping (HK) protein (beta-actin for A549 cells and tubulin for H1650 cells). Finally, HK normalized bands, were further normalized versus not-treated (NT) cells.

Western blot analysis was performed to determine STAT3 expression in vitro. HaCaT cells were treated with PBS, Dextran (20 μg/ml), ACT (20 μg/ml)or ACT-Dex NPs (20 µg ACT equiv./mL) for 12 h. Subsequently, the cells were further stimulated using recombinant IL-22 (PeproTech; 50 ng/ml) for an additional 1 h, and the cell lysate was prepared. After electrophoresis, proteins were electroeluted onto a polyvinylidenedifluoride (PVDF) membrane (Invitrogen, Carlsbad, USA). Antibodies: anti-pSTAT3 (Tyr705) (Cell Signaling, Beverly, USA), STAT3 (Cell Signaling), and anti-actin (Abcam, Cambridge, United Kingdom). For the visualization of the immunoreactive proteins, an enhanced chemiluminescence assay kit and a chemiluminescence imaging system (ChemiScope 6200T, ClinX, Shanghai, China) were used.

Tissues containing ischemic penumbra were harvested for Western blotting at 24, 48, 72 h after MCAO following a standard protocol (Molecular Clone, Edition II). Briefly, the tissues were lysed in 300 μL lysis buffer (10 mM Tris, 150 mM NaCl, 1 % Triton X-100, 0.5 % NP-40, 1 mM EDTA, pH 7.4) and mixed with protease and phosphatase inhibitor cocktails (Roche). Nuclear protein was obtained using nuclear and cytoplasmic extraction reagents (Thermo Scientific). Twenty-five micrograms of cell lysate, as quantified with a BCA protein assay (Pierce), was separated on SDS-PAGE and transferred to PVDF membranes (Roche). The primary antibodies used were as follows: anti-glial fibrillary acidic protein (GFAP) (1:4000, Cell Signaling Technology), anti-Iba1 (1:1000, Abcam), anti-DRD2 (1:2000, Abcam), anti-p-STAT3 (Tyr705) (1:1000, Cell Signaling Technology), anti-STAT3 (1:1000, Cell Signaling Technology), anti-CRYAB (1:2000, Abcam), anti-β-actin (1:2000, Abcam), and anti-histone 3 (1:2000, Cell Signaling Technology). The secondary HRP-labeled antibodies were all from Cell Signaling Technology. Chemical reactions were detected with an ECL system (Advansta), and the scanned images were analyzed with ImageJ software (version 1.47). The protocols for cell culture experiments were the same as those described above.