NK cell effector cells were co-cultured with target cells that had been pre-incubated with 100 µCi 51Cr for 1 or 4 hr in 96-well round-bottomed plates at 37°C 5% CO2. 1% IGEPAL (v/v) (Sigma-Aldrich) was used to lyse maximal release control wells and plates were centrifuged. Supernatant was transferred to a LUMA plate (Perkin Elmer) and dried overnight. Plates were read with a TopCount NXT and % specific lysis was calculated as follows: (sample – average spontaneous release) / (average total release – average spontaneous release) x 100. For experiments done with Pyk2 inhibition, effectors were pre-incubated for 10 min with 5 µM PF431396 (Tocris) then assays were performed in the presence of 5 µM PF431396 or equivalent volume of DMSO as a vehicle control.
Pf 431396
Manufactured by Bio-Techne
Sourced in United Kingdom
PF 431396 is a small molecule inhibitor developed by Bio-Techne. It functions as a selective and potent inhibitor of focal adhesion kinase (FAK). The product is intended for research use only.
Automatically generated - may contain errors
Lab products found in correlation
U0126, by Cayman Chemical (3 mentions)
Pf573228, by Bio-Techne (2 mentions)
Gkt137831, by Cayman Chemical (2 mentions)
Lumaplate, by PerkinElmer (1 mentions)
Tnp ova, by Biosearch Technologies (1 mentions)
Interleukin 3 (il 3), by R&D Systems (1 mentions)
Human il 6 quantikine elisa kit, by R&D Systems (1 mentions)
Pcdna3.1 hygro, by Thermo Fisher Scientific (1 mentions)
Peyfp n1 vector, by Takara Bio (1 mentions)
Pgex 5x 3, by GE Healthcare (1 mentions)
Pd173074, by Bio-Techne (1 mentions)
Bapta am, by Cayman Chemical (1 mentions)
Forskolin, by Merck Group (1 mentions)
Latrunculin a, by Bio-Techne (1 mentions)
8 br camp, by Abcam (1 mentions)
Carbenoxolone, by Merck Group (1 mentions)
H89 dihydrochloride, by Merck Group (1 mentions)
Ipa 3, by Selleck Chemicals (1 mentions)
Sb203580, by Selleck Chemicals (1 mentions)
Y 27632, by Selleck Chemicals (1 mentions)
11 protocols using pf 431396
1
Chromium-release Cytotoxicity Assay with Pyk2 Inhibition
2
Inhibition of PYK2 Kinase Activity
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Chemical inhibiter against PYK2 (PF-431396) (Tocris Bioscience) has been used to inhibit the kinase activity. hBMVECs were cultured till confluency and treated with 22 nM PF-431396. Cells treated with equal volume of DMSO have been taken as control. Diphenyleneiodonium chloride (DPI) (Sigma Aldrich) dissolved in DMSO has been used as ROS scavenger, at 100 nM concentration. hBMVECs were harvested for lysate preparation after 12 hours of treatment.
3
Basophil Migration Assay Protocol
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To generate a sufficient source of basophils for the assay, we administered IL-3 complexes (10 µg IL-3 [R&D Systems] complexed to 50 µg anti–IL-3 [MP2-8F8; BioLegend] for 5 min at room temperature) to Basoph8 mice 3 d before migration assays (Finkelman et al., 1993 (link)). Mice were then sensitized with 5 µg monoclonal IgE against TNP (C38-2; BD) 24 h before the assay. On the day of the migration assay, splenocytes were enumerated, and a total of 12,500 basophils were placed in the upper chamber of the transwell apparatus with LTB4 at 5 nM ± 100 ng/ml TNP-OVA (Biosearch Technologies) placed in the lower chamber analogous to published protocols (Ansel et al., 1999 (link)). For transendothelial migration assays, 1.5 × 105 bEnd.3 cells (an immortalized, mouse brain endothelial cell line) were placed on gelatinized transwell inserts 40 h before the assay (Montesano et al., 1990 (link)). Basophils in the lower chamber were assessed by flow cytometry after a 4-h incubation at 37°C. Each condition was performed in duplicate. Inhibitors PF 431396 and PF 573228 (Tocris Bioscience) were diluted to the indicated concentration and placed in both the upper and lower wells of the transwell just before addition of the basophils.
4
Quantifying IL-6 Using ELISA and PYK2 Inhibitor
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For ELISA we used a Human IL-6 Quantikine ELISA Kit from R&D systems (#S6050). ELISA was performed on cell supernatants according to the manufacturer protocol. PYK2 inhibitor (PF 431396) had been purchased in Tocris (Cat. No. 4278).
5
Molecular Cloning and Transfection Techniques
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Constructs of SKAP1 and GRB-2 were inserted into a pGEX5x-3 (GE Healthcare), into a 3xFlag-tagged and EGFP-tagged pcDNA3.1-Hygro (Invitrogen) vector. LAT and various mutants were cloned into a myc and 3xFlag-tagged pcDNA3.1 vector. PYK2 and FAK were cloned in a 3xFlag-tagged vector. SKAP1 and PLCgamma1 were cloned in pSRalpha expression vector in-frame with HA-tag. SLP-76 cDNA was sub-cloned in the pEYFP-N1 vector (Clontech). Mutants were generated by site-directed mutagenesis using the Quick Change protocol and Pfu Ultra II Fusion HS DNA Polymerase (Stratagene). All constructs were confirmed by sequencing. Transfections were performed in 4mm gap-cuvettes with the use of a BTX ECM 830 electroporator with a single pulse of 385 V and 6 ms. Cells were immediately transferred to pre-warmed complete medium and allowed to recover for 24 h prior to analysis. FAK and PYK inhibitor PF 431396 and PF 573228 were purchased from Tocris.
6
Inhibiting FGFR and FAK in Cell Cultures
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To inhibit FGFR activity, cultures were treated with 50 nM PD173074 (Tocris), which is selective for FGFRs at this concentration (IC50 values are 5 and 21.5 nM for FGFR3 and FGFR1, respectively; Tocris website). To inhibit FAK activity, cultures were treated with 10 nM PF 431396 (Tocris), which also inhibits proline-rich tyrosine kinase 2 (PYK2) (IC50 values are 2 and 11 nM respectively; Tocris website).
7
Comprehensive Reagent Acquisition Protocol
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8
Pharmacological Modulation of Hs578t Cells
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Hs578t WT and KO cells (75,000 cells/well) were seeded in a 6-well plate. Drugs were applied and the cells were incubated overnight (16 h) or for 8 h (for carbenoxolone, PF 431396, IPA-3, and latrunculin A). The final concentrations of the drugs were as follows: meclofenamate sodium (Chem Cruz; #sc200532A) 25 µM; forskolin (Sigma-Aldrich; #F6886), 30 µM; latrunculin A (Tocris; #3973, Bristol, UK), 10 nM; SB203580 (Selleckchem; #S1076, Houston, TX, USA), 25 µM; carbenoxolone (Sigma-Aldrich; #C4790), 25 µM; Y-27632 (Selleckchem; #S1049), 10 µM; IPA-3 (Selleckchem; #S7093), 2.5 µM; 8-Br-cAMP (Abcam; #ab141448, Cambridge, UK), 100 µM; H89 dihydrochloride (Sigma-Aldrich; #C4790), 20 µM; and PF 431396 (Tocris; #4278), 5 µM.
9
Collagen Microgel Contractility Assay
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Cell-laden collagen microgels were stimulated with or without 10 ng/mL TGF-β1 (R&D Systems) and anti-fibrotic drugs: nintedanib (Selleck Chem), pirfenidone (Selleck Chem), and the focal adhesion kinase inhibitor PF 431396 (Tocris) at specified concentrations. These collagen microgels were imaged over 8 days to monitor contraction. To determine the half maximal inhibitory concentration (IC50), the area under the curve (AUC) of the area over time graph was calculated for each individual gel as a parameter of overall contractility. These values were normalized, such that a gel with no contraction would have a normalized AUC of 100%. These contraction responses were fit to sigmoidal curves using the scipy module in python (Virtanen et al., 2020 (link)), using Eq. 1:
where A is the extent of contraction in the control condition, B is the Hill Coefficient, and C is the IC50.
where A is the extent of contraction in the control condition, B is the Hill Coefficient, and C is the IC50.
10
Intracellular Calcium Signaling Assay
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All chemicals or reagents were obtained from Sigma‐Aldrich, unless specified otherwise. Yoda1, PF431396, GsMTx4, and PPADS were from Tocris. U0126 and ionomycin were from Cayman Chemical. Ruthenium red (RR) was from Merck. Ca2+/Mg2+‐free phosphate‐buffered saline (PBS), Dulbecco's modified Eagle's medium (DMEM), GlutaMAX‐I, OPTI‐MEM, fetal bovine serum (FBS), penicillin‐streptomycin, trypsin‐EDTA, Fura‐2/acetoxymethyl (Fura‐2/AM), and pluronic acid F‐127 were from Invitrogen.
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