Rabbit polyclonal antibodies

Human recombinant monocyte chemoattractant protein 1 (MCP-1) protein was purchased from PeproTech (Rocky Hill, NJ, USA). Anti-rabbit and anti-mouse IgG-conjugated horseradish peroxidase, rabbit polyclonal antibodies (specific for p-GSK3β (Santa Cruz sc-135653) and GSK3β (Santa Cruz sc-9166)), and mouse monoclonal antibodies (specific for VEGF-A (Santa Cruz sc-7269), β-actin (Santa sc-47778), MCP-1 (Santa Cruz sc-32771), ILK (Santa Cruz sc-20019), p-MEK1/2 (Santa Cruz sc-271914), MEK1/2 (Santa Cruz sc-6250), p-ERK1/2 (Santa Cruz sc-7383), and ERK1/2 (Santa Cruz sc-1647)) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The control miRNA and miR-29c mimic were purchased from Life Technologies (Carlsbad, CA, USA). Lipofectamine 2000 was purchased from Invitrogen (Carlsbad, CA, USA). All other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA).

The allosteric modulator LUF6000 (N-(3,4-dichloro-phenyl)-2-cyclohexyl-lH-imidazo [4,5-c]quinolin-4-amine) was synthesized for Can-Fite BioPharma at Leiden Academic Centre for Drug Research (Leiden, The Netherlands)/Haoyuan Chemexpress Co., Ltd (Shanghai, China). A stock solution of 10 mM was prepared in DMSO and further dilutions were prepared in PBS. ConA (Canavalia ensiformis, Jack Bean Hemagglutinin) was purchased from Calbiochem-EMD Millipore (San Diego, CA). Monosodium iodoacetate (MIA; Sigma, St. Louis, MO) was prepared in saline solution.
Rabbit polyclonal antibodies against rat A₃AR, phosphoinositide 3-kinase (PI3K), IκB kinase (IKK), IκB, nuclear factor kappa B (NF-κB), Janus kinase 2 (Jak-2), and signal transducer and activator transcription 1 (STAT-1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and β-actin were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).

The protease inhibitor mixture ethylenediaminetetraacetic acid (EDTA) was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). 2′,7′-Dichlorofluorescein diacetate (DCF-DA) was obtained from Molecular Probes (Eugene, OR, USA). Rabbit polyclonal antibodies against peroxisome proliferator-activated receptor α (PPARα), sterol regulatory element binding protein-1 (SREBP-1) and sterol regulatory element binding protein-2 (SREBP-2), 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR), and mouse monoclonal antibodies against fatty acid synthase (FAS), β-actin, and histone, and goat polyclonal antibody against mitochondrial uncoupling protein 2 (UCP-2) and stearoyl-CoA desaturase-1 (SCD-1) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Rabbit polyclonal antibodies against acetyl-CoA carboxylase (ACC), AMPKα, and phosphor-AMPKα were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). ECL Western Blotting Detection Reagents and nitrocellulose membranes were supplied by GE Healthcare (Buckinghamshire, UK).

The localization of hsa-miR-212-3p and RFXAP in pancreatic cancer tissue was determined by fluorescence in situ hybridization and immunohistochemistry, respectively. In the present study, 20 paired tissues were assessed. All patients provided their written informed consent prior to recruitment.
In situ detection was performed using 4-um sections of pancreatic ductal adenocarcinoma tissue with a FITC-labeled LNA oligonucleotide probe for miR-212-3p. The probe was denatured at 73°C and hybridized overnight at 37°C. The procedure was carried out according to manufacturer's instructions. Nuclei were stained with 4,6-diamino-2-phenylindole (DAPI; Sigma, USA). For imaging and processing, a conventional epifluorescence microscope equipped with FITC and DAPI filters (Nikon, Japan) and Image-Pro Plus software was used, respectively.
Immunohistochemical staining was carried out using standard streptavidin-biotin-peroxidase complex method. Briefly, paraffin-embedded tissue sections were dewaxed and rehydrated through an alcohol series, and then endogenous peroxidase activities were blocked. After non-specific sites were saturated with a rabbit polyclonal antibodies(Santa Cruz Biotechnology Inc Santa Cruz, CA, USA), slides were incubated overnight at 4°C with RFXAP antibody (1:100, #ab172281, abcam). Sample incubated with PBS instead of primary antibody were used as negative control.

Extracted RNA was reverse transcribed in a thermal cycler using RT enzyme. The real-time PCR was carried out in ViiA 7 system using TaqMan® gene expression assays. The cycling conditions were 50 °C for 2 min and 95 °C for 10 min, then 40 cycles of 95 °C for 15 s and 60 °C for 1 min. The comparative ΔΔCt method was applied to calculate fold change. For endogenous controls, GAPDH was used for Ahr, CYP1A1 and SOX4, and RNU6B was used for miR-212/132. System, reagents and kits for real-time PCR were all from Applied Biosystems, Grand Island, NY. For western blot, cell were lysed by RIPA lysis buffer system (Santa Cruz Biotechnology), lysate was then fractionated using SDS-PAGE system (Bio-Rad, Richmond, CA). The Ahr, CYP1A1, SOX4 and β-actin were detected using their rabbit polyclonal antibodies (Santa Cruz Biotechnology). The intensities of the protein bands were quantified via software [http://imagej.nih.gov/ij/download.html] ImageJ v.1.48 [52 (link)].