Antibody against β actin

As previously described [55 (link), 56 (link)], antibodies against FABP5 were obtained from R & D systems and β-tubulin was purchased from Sigma Aldrich Co (St. Louis, MO). Antibodies for PPARβ/δ and p65 were obtained from Santa Cruz (Santa Cruz, CA) and Cell Signaling (Boston, MA), respectively. Use of PPARβ/δ and p65 antibodies was referenced in [57 (link), 58 (link)], respectively. Antibody against β-actin was purchased from Cell Signaling (Boston, MA). Anti-mouse and anti-rabbit immunoglobulin horseradish peroxidase-conjugated antibodies were from BioRad, and anti-goat immunoglobulin was from Santa Cruz. Curcumin (C-1386) and all-trans-retinoic acid (R-2625) were obtained from Sigma. MTT reagent (3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) was purchased from Sigma.

Western blot analysis was carried out as described previously [20 (link)]. Total protein was extracted from BV2 microglia. Equal amounts of protein were separated on sodium dodecyl sulfate polyacrylamide gels, transferred to polyvinylidene fluoride membranes, and blocked with 5% bull serum albumin for 1 h at 25 °C. Membranes were incubated overnight with an antibody against iNOS (1:800, Abcam, Inc., Boston, MA, USA), an antibody against ARG1 (1:800, Abcam, Inc., Boston, MA, USA), an antibody against CD206 (1:1200, Abcam, Inc., Boston, MA, USA), or an antibody against β-actin (1:1200, Cell Signaling Technology, Inc., Danvers, MA, USA), then washed and incubated with horseradish peroxidase-coupled secondary antibodies for 2 h at 25 °C. The protein bands were detected by chemiluminescence (BioRad, Inc., Hercules, CA, USA), and their optical density was measured using Quantity One software (BioRad, Inc., Hercules, CA, USA). Relative protein levels were normalized to β-actin.

Replicate cultures of 1 × 10⁶ RAW 274.7 cells per well were seeded in 6-well plates, then treated with RAP (50 μg/mL) with/ without Taxol (10 μM) for 24 h. After that, cells were washed twice with phosphate buffered saline and harvested in cold lysis buffer containing protease inhibitors or phosphatase inhibitors. Cell lysates were collected from culture plates, and protein were collected by centrifugation. The concentrations of proteins were determined by a BCA protein assay (Pierce Biotechnology, Rockford, IL, United States). The total protein was boiled in 4× loading buffer (Bio-Rad, United States) for 10 min at 100°C, then loaded into various concentrations of Tris–HCl-Polyacrylamide gels according to its molecular properties (from 6 to 12%), and transferred electrophoretically to an Immobilon-P membrane (Millipore Corporation, Billerica, MA, United States). Membranes were incubated with primary antibodies and appropriate horseradish peroxidase-labeled secondary antibodies. Membranes were additionally probed with an antibody against β-actin (Cell Signaling) to normalize the loading of proteins among samples. In the last step, secondary antibodies were detected by chemiluminescent agents (Pierce Biotechnology).

Dulbecco’s modified Eagle’s medium (DMEM) and Roswell Park Memorial Institute (RPMI) 1640 medium were obtained from Nacalai Tesque (Kyoto, Japan). Fetal bovine serum (FBS; #172012) was purchased from Sigma Aldrich (St. Louis, MO). Antibody against CD63 (#11–343-C025) was purchased from EXBIO (Prague, Czech Republic). Donkey anti-mouse IgG 10 nm gold (#ab39593) was purchased from Abcam (Cambridge, UK). Growth factor-reduced basement membrane protein (Matrigel; #356230) was purchased from Corning (New York, NY). Antibodies against vitronectin 65/75 (D-8; #sc-74,484) and fibronectin (EP5; #sc-8422) were purchased from Santa Cruz Biotechnology (Dallas, TX). Antibody against β-actin (#4967) was purchased from Cell Signaling Technology (Danvers, MA).

The protein extracts from cultured cells were homogenized in radioimmunoprecipitation assay lysis buffer (50 mM Tris–HCl, pH 8.0, 150 mM NaCl, 5 mM EDTA, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, and 1:100 protease inhibitor cocktail). Protein concentrations were determined using the bicinchoninic acid assay (Pierce) and denatured in the protein loading buffer. Equal amounts of protein were resolved on 4–12% Bolt^Tm Bis–Tris plus gel, and immunoblot analyses were performed using antibodies against C3 (1:1000; Millipore). An antibody against β-actin (1:5000; Cell Signaling Technology) was included as an internal standard to monitor loading errors.

Pterostilbene 4′-β-glucoside (4-PG) was donated from Okayama University of Science, Dept. of Life Science. Pterostilbene (PETR) was purchased from Tokyo Chemical Industry (TCI), Japan. 4-PG and PTER were dissolved in DMSO, as described previously [14 (link)]. Lipopolysaccharide (LPS), carbon monoxide-releasing molecule- (CORM-) 2, bilirubin, and ammonium iron(II) sulfate hexahydrate were purchased from Sigma-Aldrich, (St. Louis, MO, USA). Antibody against HO-1 was purchased from Enzo Life Sciences (Farmingdale, NY, USA), and antibody against β-actin was purchased from Cell Signaling (Danvers, MA, USA). siRNA against HO-1 was from Santa Cruz Biotechnology (Santa Cruz, CA). Zinc protoporphyrin IX (ZnPP) was from Frontier Scientific (Logan, UT, USA).