Ab62331

Mouse cerebellar tissues and B-lymphocyte cell pellets were homogenized with a cell lysis buffer (Cell Signaling) with Halt phosphatase inhibitor (Thermo-Fisher), complete protease inhibitors (Roche) and PSMF (Sigma-Aldrich). Thirty micrograms of lysates were loaded into 4–12% Bis–Tris gels (Invitrogen). Electrophoresis was carried out according to the manufacturer's recommendations. Following electrophoresis, the proteins were transferred to nitrocellulose membranes by the iBlot device (Invitrogen), blocked with an Odyssey blocking buffer (LI-COR Biotechnology) for 1 h. Membranes were incubated overnight with the following primary antibodies in blocking buffer: COX1 (ab133319 for human, ab133319 for mouse; Abcam), COX2 (ab62331 for human, ab6665 for mouse; Abcam), phospho-CREB (4095), phospho-cJun/AP1 (5464), phospho-NFκB (4025; Cell signaling), cPLA2 (sc-454), phospho-cPLA2 (sc-34391; Santa Cruz Biotech, Santa Cruz, CA, USA), β-actin (A5441; Sigma-Aldrich), and β-tubulin (DSHB-E7; DSHB, Iowa). Subsequently, the membranes were incubated with a corresponding pair of IRDye 680CW and IRDye 800CW-coupled secondary antibodies (LI-COR). Proteins were visualized with the Odyssey infrared imager and software (LI-COR) according the manufacturer's instruction.

Freshly isolated retinal tissues were homogenized and lysed in RIPA buffer (P0013; Beyotime, Beijing, China). The protein concentration was quantified by a Bicinchoninic Acid kit (Pierce, Rockford, IL, USA). Western blotting was done as described in our previous study (20 (link)) with the following antibodies: anti-PPARα (ab215270; Abcam; 1:1,000), anti-GFAP (ab33922; Abcam; 1:1,000), anti-COX2 (ab62331; Abcam; 1:1,000), and anti-β-actin (GB12001; Servicebio; 1:3,000). The expression levels of PPARα, GFAP and COX2 were quantified by Image-Pro Plus 6.0 (Media Cybernetics, Rockville, MD, USA) and normalized by the densitometry of β-actin.

Cell lysis was performed with RIPA buffer (P0013D; Beyotime, Shanghai, China) supplemented with protease (Selleck, Shanghai, China) and phosphatase inhibitors (Selleck). The BCA Protein Assay Kit (PC0020; Solarbio, Beijing, China) was used to measure protein concentrations. The samples were boiled at 100 °C and centrifuged to obtain the supernatant, after which 100 µg of protein was loaded onto 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis gels (EpiZyme, Shanghai, China). The proteins were transferred to 0.45 µm polyvinylidene fluoride (PVDF) membranes (Millipore, Burlington, MA, USA), and the membranes were blocked in a Western blocking buffer (P0023B; Beyotime) for 2 h at 15 °C. The sections were then incubated with primary antibodies (1:2000) overnight at 4 °C and with the relevant secondary antibody (1:10,000) for 2 h at 15 °C. Bands were visualised using BeyoECL Plus (P0018S; Beyotime). Protein bands were quantified relative to the loading control (GAPDH). The following antibodies were used for Western blotting: anti-GAPDH (AF1186; Beyotime), anti-rabbit-IgG-HRP (A120-111P; Bethyl, Montgomery, TX, USA), and anti-COX2 (ab62331; Abcam, Cambridge, UK).

Primary antibodies against, non-phospho (active) β-Catenin (Ser33/37/Thr41, #8814), CHOP (#2895), AXL (#8661), ABCG2 (#4477), E-cadherin (#3195), cleaved caspase-9 (Asp315, #9505), and cleaved caspase-3 (Asp175, #9664) were purchased from Cell Signaling (Danvers, MA, USA). Primary antibodies for CD133 (ab19898), CD44 (ab51037), cleaved caspase-4 (ab75182), COX-2 (ab62331), vimentin (ab92547), N-cadherin (ab76011), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (ab8245) were purchased from Abcam (Cambridge, MA, USA). The primary antibody for GRP78 (#PA1-014A) was purchased from Thermo Fisher Scientific™ (Waltham, MA, USA). The primary antibody for p-MET (sc-34086) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Total protein was extracted from tumor tissue in the spleen as described previously [15 (link)], and protein concentration was determined by BCA protein assay kit (Beyotime, P0012). Western blotting was performed with the following primary antibodies: anti-IL–6 (1:500, Abcam, ab154367), anti-VEGF (1:1000, Abcam, ab46154), anti-MMP–9 (1:500, Abcam, ab38898), anti-TGF-β(1:1000, Abcam, ab31013), andanti-PTGS2 (1:500, Abcam, ab62331), followed by incubation with anti-rabbit horseradish-peroxidase-conjugated secondary antibody (1:1000). Proteins were visualized by an ECL chemiluminescence detection system. Detection of GAPDH (1:2000, Abcam, ab181602) served as internal loading control.

Protein expression was examined by Western blot as previously described [34 (link)]. HPMECs or lung tissues were lysed using RIPA buffer, and protein concentration was examined using the Pierce BCA protein assay kit (23227, Thermo Fisher Scientific, USA). The polyvinylidene difluoride (PVDF) membranes were purchased from GE Healthcare (10600023, USA). The following primary antibodies were employed: VE-cadherin (1 : 1000, ab33168, Abcam, USA), β-catenin (1 : 1000, 610154, BD Transduction Laboratories, USA), dephosphorylated active β-catenin (05-665, Millipore, Germany), Sirt3 (1 : 500, ab189860, Abcam, USA), matrix metallopeptidase-7 (MMP-7, 1 : 400, ab5706; Abcam, USA), and cyclooxygenase-2 (COX-2, 1 : 1000, ab62331, Abcam, USA). α-Tubulin was purchased from the Proteintech Company (Hubei, China). The secondary antibodies of goat antirabbit (1 : 5000, ab6721; Abcam, USA) or goat antimouse (1 : 5000, A21010; Abbkine, USA) were used. PVDF membranes were visualized using a chemiluminescence Western blotting detection reagent. The signal intensity of each immunoblot was analyzed using ImageJ software (version 1.48v; NIH, USA), and each band density was normalized by the α-tubulin protein expression level. To clearly present the results, the value of protein expression in the sham-operated or control group was further normalized to 1 using the mean.

After 2 days of IR or SHAM procedure, the rats were euthanized by an overdose of
the anesthetic (thiopental 100 mg/kg) and renal tissue samples were collected,
fixed in a 4% paraformaldehyde in PBS 0.1 M (pH 7.4) for 24 h at 4°C, and
embedded in paraffin. The study was performed by immunohistochemistry method as
described before15 (link)
^-16 (link) with immunoperoxidase reaction. The
tissue fragments were incubated overnight at 4°C with primary antibodies. The
inflammation was studied using anti-COX-2 (1:500, ab62331, ABCAM, Cambridge, UK)
and anti-TGF-β (1:80, SC-7892 policlonal, Santa Cruz, CA, USA) markers. The
markers for apoptosis and injury were anti-caspase-3 (1:1000, 9662, Cell
Signaling Technology, Danvers, MA, USA) and anti-vimentin (1:500, M0725, Dako,
Denmark). Twenty-five fields of the renal juxtamedullary region were evaluated
in one slide from each animal to obtain an average of the scores performed as
previously described.14 (link)
^-16 (link)