Recombinant human cxcl12

Recombinant human CXCL12 and IL-21 were purchased from Peprotech (Rocky Hill, NJ). Peptidoglycan (PGN) poly (I:C), LPS, and R848 were purchased from Sigma-Aldrich (Poole, Dorset, UK). Human CpG-B DNA was purchased from Hycult Biotech (Uden, Netherlands). Flagellin was provided by Dr. Myoung Ho Jang (Osaka University). RPMI 1640 medium and fetal bovine serum (FBS) were purchased from Gibco BRL (Eggenstein, Germany). Anti-ERK1/2 and anti-human phospho-AKT1/2/3 monoclonal antibodies (Ab) were obtained from Cell Signaling Technology. Anti-ß-actin and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) Abs were obtained from Santa Cruz (Paso Robles, CA). Anti-RGS1 Ab was purchased from Novus Biologicals (Littleton, CO). The anti-CXC chemokine receptors (CXCR) 4-APC, TCRαβ-FITC, CD38-PerCP-Cy5.5, and CD20-APC were purchased from BD Biosciences (Heidelberg, Germany). The BCA protein reagent was purchased from Thermo Scientific (Hudson, NH). Interleukin (IL)-2 and IL-4 were provided by Prof. Jongseon Choe (College of Medicine, Kangwon National University).

Recombinant human CXCL12 was from Peprotech (London, UK), synthetic analogue [N33A]CXCL12 was from Peptidesynthetic (Fareham, UK), human synthetic CXCL12 COOH-terminally conjugated with Alexa Fluor 647 (CXCL12^AF647) was obtained from Almac Sciences (Craigavon, UK).

The human PCa cell lines CFPAC-1, BxPC-3, MiaPaCa-2, AsPC-1, SW1990, Panc-1, and the rat Schwann cells RSC96 were purchased from the Chinese Academy of Sciences Cell Bank of Type Culture Collection (CBTCCCAS). According to the instructions, all cell lines were cultured in the proper medium (HyClone, Logan, USA) supplemented with 10% fetal bovine serum (FBS), 100 μg/ml ampicillin and 100 μg/ml streptomycin. The cultures were incubated at 37°C in a humidified atmosphere containing 5% CO₂.
Recombinant human CXCL12 was purchased from PeproTech (Rocky Hill, USA). The pharmacological reagent AMD3100 was purchased from Sigma (St. Louis, MO, USA). Antibodies were purchased from the following sources: anti-CXCR4, anti-CXCL12, anti-NGF, anti-β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-MMP-2, anti-uPA (Bioworld, Minneapolis, MN, USA), anti-S100 and anti-NF200 (Abcam, USA).

MNP-treated and -untreated murine NK cells expanded in vitro in the presence of IL-2 or the human NK-92MI cell line were differentially labeled with PKH26 Red or PKH67 Green fluorescent cell-linker kits (Sigma-Aldrich) and mixed at a 1:1 ratio, after which 5 × 10⁵ cells were seeded in 0.1 ml of the appropriate medium in a transwell insert (Corning, 5-μm pore). The chemotactic gradient was created by adding the recombinant murine fractalkine (CX3CL1, aa25-105) (100 ng/ml, R&D Systems) for murine NK cells or the recombinant human CXCL12 (100 ng/ml, Peprotech) for human NK-92MI to the lower chamber. The cells migrated for 16 h, after which cells from lower chambers were counted by flow cytometry. Cell migration was quantified and normalized for loading into an input well. In some cases, the magnet B was placed below the well.

Cdk5 (Abcam, Cambridge, UK; clone 2G2), cytokeratin 5 (Abcam, EP1601Y), cytokeratin 8 (BioLegend, London, UK; clone 1E8), drebrin (GeneTex, Isleworth, UK; GTX11068), drebrin (Abcam 176318 & 178408), drebrin (Progen, Heidelberg, Germany; clone Mx823), EB1 (BD Transduction Laboratories, Oxford, UK; clone 5), EB3 (Millipore, Consett, UK; AB6033), p35 (Abcam, ab66064), GAPDH (GeneTex, clone GT239), GFP (Abcam, 6556), laminin (Sigma-Aldrich, Poole, UK; clone LAM-89), p35/25 (Cell Signaling, Hitchin, UK; C64B10), p63 (Abcam, ab735 clone BC4A4), pERK1/2 (Thr202/Tyr204) antibody (Cell Signaling; #9101), pS142-drebrin (Millipore, clone 3C14), tyrosinated α-tubulin (SeroTec, Kidlington, UK; clone YL 1/2), vimentin (Dako, Glostrup, Denmark; clone V9). Horseradish peroxidase-conjugated secondary antibodies were from Dako. Alexa-conjugated secondary antibodies and phalloidin were from Life Technologies, Warrington, UK. BTP2 (YM-58483) was from Cambridge Bioscience (Cambridge, UK; CAY13246). Recombinant human CXCL12 was from PeproTech (London, UK; 300-28A). Matrigel was from Corning (New York, USA; 354234) and fibronectin from Millipore.