Nf κb pathway sampler kit

Recombinant mouse soluble RANKL (Gene ID: 21943) was obtained from R&D Systems (Catalog number: 462-TEC-010, Oakville, ON, Canada). Dulbecco modified Eagles medium (DMEM), fetal bovine serum (FBS), penicillin-streptomycin (10,000 U/mL), and TRIzol Reagent were purchased from Thermo Fisher Scientific (Burlington, ON, Canada). Ovotransferrin (conalbumin from chicken egg white) with purity ≥98% was purchased from Sigma-Aldrich (Catalog number: C0755, St. Louis, MO, USA). The tartrate-resistant acid phosphatase (TRAP)-staining kit was obtained from Sigma-Aldrich (Catalog number: 387A-1KT, St. Louis, MO, USA). The annexin V-FITC Apoptosis Staining/Detection Kit was purchased from Abcam (Catalog number: ab14085, Toronto, ON, Canada). The NF-κB pathway sampler kit (Catalog number: 9936T), MAPK family antibody sampler kit (Catalog number: 9926T), and phosphor-MAPK family antibody sampler kit (Catalog number: 9910T) were purchased from Cell Signaling Technology (Whitby, ON, Canada). Recombinant anti-TRAF6 antibody (Catalog number: ab33915), anti-c-Fos antibody (Catalog number: ab190289), and anti-α tubulin antibody (Catalog number: ab7291) were purchased from Abcam (Cambridge, MA, USA). NFATc1 antibody (Catalog number: 7A6) and cathepsin K antibody (Catalog number: E-7) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

NF-κB Pathway Sampler Kit, Akt, p-Akt (Ser 308), p-Akt (Thr 473), p21, p-CDK2, CDK2, cyclinA, cyclinD1, mTOR Pathway Sampler Kit, and Apoptosis sampler Kit were obtained from Cell Signaling Technology (Beverly, MA, USA). All the cell lines were treated with NVP-Bez235 and lenalidomide in single way or combination for 72 h. Bortezomib was purchased from Selleck (Huston, USA). Cells were performed with 5 nM Bortezomibe for 24 and 48 h, respectively. Then the samples in each group were collected and separated by standard SDS-PAGE gel electrophoresis and transferred to NC membrane. Blocked with 5 % non-fat milk supplemented in 0.1 % TBST, membrane was probed with primary antibodies at 4 °C overnight. After washed with TBST, membrane was incubated with HRP-conjugated secondary antibodies (Santa Cruz, CA) for one hour. The bands were detected and quantified in Image Lab software (Bio-Rad Laboratories, California, USA).

Following treatment, cells were lysed, and total protein concentration was measured by colorimetry using a BCA kit (Thermo Fisher Scientific, catalog 23225), after which 25 μg of protein was separated by 4%–15% mini-Protean TGX gel (Bio-Rad) and transferred to a nitrocellulose membrane (Bio-Rad) using Trans-Blot Turbo System (Bio-Rad). Proteins underwent IB with the following Abs: JAK3 (Cell Signaling Technology, catalog 8827S), STAT1 (Cell Signaling Technology, catalog 9172S), p-STAT3 (Cell Signaling Technology, catalog 9131S), STAT5 (Cell Signaling Technology, catalog 94205S), STAT6 (Cell Signaling Technology, catalog 5397S), SOCS2 (Cell Signaling Technology, catalog 2779S), SOCS6 (Abcam, catalog ab197335), NF-κB Pathway Sampler Kit (Cell Signaling Technology, catalog 9936), and GAPDH (Cell Signaling Technology, catalog 5174S). Primary Abs were detected by binding goat anti-rabbit IgG–HRP (Cell Signaling Technology, catalog 7074) and visualized by the enhanced chemiluminescence method (Thermo Fisher Scientific, catalog 32209).

Phorbol-12-myristate-13-acetate (PMA), gelatin, Triton X-100, Coomassie brilliant blue R-250 was purchased from Sigma-Aldrich, MMP-9 inhibitor (SB-3CT) and NF-κB inhibitor (sc-75741) were purchased from Selleck. Recombinant human MMP-9 protein and Recombinant mice MMP-9 protein were purchased from R&D systems. Recombinant human pro-MMP-9 protein were purchased from ProSpec. Commercialized DENV2-NS1 protein were purchased from Native Antigen. Trizol reagent was purchased from Ambion. Lipofectamine 2000 reagent was purchased from Invitrogen. Human MMP-9 ELISA kit was purchased from BD Biosciences. Membrane maker Dil (D8700) was purchased from Solarbio. NF-κB Pathway Sampler Kit (#9936T), Tight Junction Antibody Sampler Kit (#8683T), Cadherin-Catenin Antibody Sampler Kit (#9961T), and Antibodies against TIMP-1 (8946S) were purchased from Cell Signaling Technology. Antibody against DENV-NS3 (GTX124252) were purchased from Genetex. Antibodies against DENV2-NS1 were purchased from Arigo biolaboratories (SQab1501). Antibodies against Flag (F3165) and HA (H6908) were purchased from Sigma. Anti-β-actin antibody (66009) were purchased from Proteintech. Rabbit IgG and Mouse IgG were purchased from Invitrogen. Anti-Mouse IgG Dylight 649, Anti-Mouse IgG Dylight 488, Anti-Rabbit IgG Dylight 649, and Anti-Rabbit IgG FITC were purchased from Abbkine.

Total proteins were extracted from hippocampus tissues or NSCs using RIPA lysis buffer (Beyotime) supplemented with phosphatase and protease inhibitors and PMSF, and then quantified by BCA assay (Meilunbio). Western blot was performed as previously described.^[24] GRP78 (#3711; 1:1000), PERK (#3192; 1:1000), p‐eIF2α (#9721; 1:1000), CHOP (#2895; 1:1000), IRE1α (#3294; 1:1000), p‐IRE1α (#9721S, 1:1000) and NF‐κB pathway sampler kit (#9936) were from Cell Signaling Technology; ATF6 (#37149, 1:1000) was from Abcam. β‐actin (MA5‐15739, 1:5000) was from Invitrogen; GAPDH (#60004, 1:5000) was from Proteintech. The horseradish peroxidase‐conjugated anti‐rabbit IgG (SA00013‐3, Proteintech) or anti‐mouse IgG (SA00001‐2, Proteintech) secondary antibodies were used, followed by detection with the enhanced chemiluminescence system (GE Healthcare). ECL Western blot analysis system was utilized for detection of the immunoreactive bands according to the manufacturer's instructions by using the GeneGnome system (Syngene). β‐actin or GAPDH was used as a loading control.

The following monoclonal or polyclonal antibodies (mAbs or pAbs) were purchased from Cell Signaling Technologies (CST, Danvers, MA, USA) and used for immunoblotting: rabbit anti-iNOS mAb (#13,120) and anti-COX-2 mAb (#12,282), NF-κB pathway sampler kit (#9936) containing two anti-mouse mAbs against I kappa B kinase (IKK) α, IκBα and five anti-rabbit mAbs against IKKβ, phospho-IKKα/β, phospho-NF-κB-p65, NF-κB-p65. Rabbit anti-GAPDH mAb (#5174) was used as loading control. Horseradish-peroxidase-conjugated anti-rabbit or anti-mouse IgG mAbs (CST) were used to visualize the proteins on the immunoblots. The water-soluble soybean-derived isoflavone glycosides (SIFs; Soyaflavone HG; Lot. 181124-02) were donated by Fuji Oil Co, Ltd. (Tokyo, Japan). The total SIF content was 51.94% (w/w) isoflavones (22.00%, as aglycone equivalents), the isoflavone composition as aglycone equivalents was described as Table 1. The proximate composition of SIF was (w/w%): moisture, 2.4%; protein, 12.2%; fat, <0.1%; minerals: 3.4%; carbohydrate, 82.0%. The energy value of the compound was 377 kcal per 100 g. dextran sulfate sodium salt (DSS; 0216011050, colitis grade) was purchased from MP Biomedicals (Santa Ana, CA, USA). Lipopolysaccharide (LPS; from E. coli serotype O55:B5, phenol extraction) was obtained from Sigma Aldrich (Darmstadt, Germany).