Anti nf κb p50

For immunostaining study, AHNP (S-NSC) cells were grown on glass cover slip before infected with Toxoplasma gondii. 24 hours after infection, cells were fixed with paraformaldehyde and standard immunefluroescent staining protocol was adopted for studying the localization of targets of interest, including NFκB, STAT3, tyrosine hydroxylase, and dopamine. Antibodies utilized include: anti-NFκB p50 (Santa Cruz Biotech, SC-7178); P-STAT3 (Y705), cell signaling (#91315); anti-tyrosin hydroxylase (Sigma, T1299); and dopamine antibody (Novus Biologicals, NB120-1001).

DHTS (PubChem CID: 11425923) was purchased from Xi’an Honson Biotechnology (Xi’an, China), and the purity was >90% according to a high-performance liquid chromatographic analysis. A stock solution of DHTS was prepared in dimethyl sulfoxide (DMSO) and stored at −20 °C. Anti-Bad, anti-Bcl-2, anti-Bcl-x_L, anti-caspase-3, anti-caspase-8, anti-caspase-9, anti-PARP, anti-phospho-JNK, and anti-phospho-p38 antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA); anti-NF-κB p50, anti-NF-κB p65, anti-p-I-κB, and anti-Bim antibodies were purchased Santa Cruz Biotechnology (Santa Cruz, CA, USA); anti-phospho-ERK and anti-FasL antibodies were purchased from BD Biosciences Taiwan (Taipei, Taiwan); anti-Bax and anti-GAPDH antibodies were purchased from GeneTex International (Hsinchu City, Taiwan); anti-Fas blocking antibody was purchased from MBL International (Woburn, MA, USA); the anti-α-tubulin antibody was purchased from Zymed Laboratories (South San Francisco, CA, USA).

Human embryonic kidney T-REx-293/hemagglutinin (HA)-MCPIP1 and T-REx-293/HA-MCPIP1-D141N cell lines were kindly provided by Dr. Yi-Ling Lin (Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan). The cells can be induced by tetracycline (Tet) to express HA-MCPIP1 or HA-MCPIP1-D141N and were cultured in Dulbecco’s modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS). Anti-cleaved caspases-3, -7, -8, and -9 as well as anti-phospho-extracellular signal-regulated kinase (ERK), anti-phospho-JNK, anti-phospho-p38, anti-phospho-IκBα, anti-phospho-IKKα/β, anti-IκBα, anti-cFLIP, anti-cIAP1, anti-HA, and anti-poly(ADP ribose) polymerase (PARP) antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA); the anti-GAPDH, anti-KPNA3, anti-KPNA4, and anti-MCPIP1 antibodies were obtained from GeneTex International (Hsinchu City, Taiwan); the anti-IKKα/β, anti-NF-κB p50, and anti-NF-κB p65 antibodies, and Protein A/G PLUS-Agarose were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA); and the anti-α-tubulin antibody was obtained from Sigma-Aldrich (St. Louis, MO, USA).

Single-stranded oligonucleotides were synthesized by Eurofins MWG Operon and annealed.
Overnight resting and anti-CD3/anti-CD28 stimulated CD4⁺ T cells from wt or Cbl-b- deficient mice were lysed and nuclear extracts were prepared. The following oligonucleotides were used, and the core binding motifs are underlined:
Binding reactions and supershifts were performed for 30 min at 4–8°C using the Binding Buffer B-1 (Active Motif 37480) together with Stabilizing Solution D (Active Motif 37488) containing 4 μg mouse T cell nuclear extracts (Active Motif 36042) and 2 μg of anti NF-κB (p50) (Santa Cruz-1190) antibody.
Binding reactions with the 3 × 10⁵ cpm labeled probe were performed for 20 min at 4°-8°C using Binding Buffer C-1 (Active Motif 37484) together with Stabilizing Solution D (Active Motif 37488). Samples were run on a 4% native polyacrylamide gel in 0.5 × TBE for 3 h at 250 V. For competition assays, 10-fold unlabeled oligonucleotides identical to the radioactive-labeled probes were added to the binding reaction.

The HeLa, C33A, and Caski cell lines were obtained from American Type Culture Collection (ATCC) and were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum at 37°C, 5% CO₂. Anti-MMP2, MMP9, TIMP-1, TIMP-2, β-actin,p38 and p-p38 were obtained from Cell Signaling Technology (Beverly, MA). Anti-NF-κB-p50, p65/Rel A and Histone H1 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) and all other chemicals were obtained from Sigma (St Louis, MO), unless otherwise indicated.

Dorsolateral prostate tissue from treated and control groups were subjected to preparation of total tissue lysate and isolation of cytosolic and nuclear fractions as described previously [38 (link)]. For Western blotting, 25 μg of protein was resolved over 4–20% Tris-glycine polyacrylamide gel and then transferred onto the nitrocellulose membrane. The blots were blocked using 5% non-fat dry milk and probed using appropriate primary antibodies overnight at 4°C. The antibodies used were anti-IKKα (sc-7218), anti-IKKβ (sc-34673), anti-NF-κB/p65 (sc-8008), anti-NF-κB/p50 (sc-8414), anti-IκBα (sc-1643) anti-p-IκBα (sc-8404), anti-Bax (sc-493), anti-Bcl2 (sc-7382), anti-COX-2 (sc-795), anti-cyclin D1 (sc-533), anti-PCNA (sc-56), and anti-VEGF (sc-152) procured from Santa Cruz Biotechnology, Santa Cruz, CA. Antibodies including anti-cleaved caspase-3 (#9661), anti-Bcl-xL (#2762), anti-p-IKKα/β (#2078) were purchased from Cell Signaling Technology, Danvers, MA. Anti-β-actin (#A1978) was obtained from Sigma-Aldrich, St. Louis, MO. The membrane was then incubated with appropriate secondary mouse (sc-2005) and rabbit (sc-2004) antibody horseradish peroxidase conjugate (Santa Cruz Biotechnology) followed by detection using chemiluminescence ECL kit (GE Healthcare Biosciences). For equal loading of proteins, the membrane was probed with appropriate loading controls.