The anti-KIR2DL4 agonistic antibody (mouse monoclonal IgG, Clone; 181703) was purchased from R&D Systems (Minneapolis, MN). Another anti-KIR2DL4 antibody (rabbit IgG, polyclonal, ab154386) and an anti-phospho-SHP-2 antibody (Y542, rabbit polyclonal IgG), used for immunostaining and Western blotting were obtained from Abcam (Cambridge, MA). An anti-phospho-ERK antibody and an anti-GAPDH antibody were obtained from Cell Signaling Technology (Beverly, MA). The KIR2DL4-targeted shRNA lentiviral particles and the control particles were purchased from Santa Cruz Biotechnology (San Diego, CA). Infection of these viral particles and the following selection were performed according to the manufacturer's instructions. The specific MAP2K1 inhibitor U0126, the specific ERK inhibitor FR180204, and the specific SHP-2 inhibitor PHPS1 were purchased from Santa Cruz Biotechnology [22 (link)–24 (link)]. We used all inhibitors at concentrations of 10 µM.
Fr180204
Manufactured by Santa Cruz Biotechnology
FR180204 is a chemical compound produced by Santa Cruz Biotechnology. It is a small molecule that can be used as a research tool in scientific studies. The core function of FR180204 is to inhibit the activity of the ERK1/2 protein kinases.
Automatically generated - may contain errors
Lab products found in correlation
Afuresertib, by Merck Group (3 mentions)
Bafilomycin, by Merck Group (3 mentions)
Anti igm f ab 2 abs, by Southern Biotech (3 mentions)
Bortezomib, by Selleck Chemicals (3 mentions)
Actinomycin d, by Merck Group (3 mentions)
Cycloheximide (chx), by Merck Group (3 mentions)
Automacs, by Miltenyi Biotec (3 mentions)
Magnetic bead, by Miltenyi Biotec (3 mentions)
Rapamycin, by Merck Group (3 mentions)
Goat anti mouse igg alkaline phosphatase, by Merck Group (2 mentions)
Goat anti rabbit igg peroxidase, by Merck Group (2 mentions)
Hrp conjugated donkey anti goat igg, by Jackson ImmunoResearch (2 mentions)
C kit, by Agilent Technologies (2 mentions)
Von willebrand factor vwf, by Agilent Technologies (2 mentions)
Wbb6f1 kit w v, by Jackson ImmunoResearch (2 mentions)
Mouse c kit neutralizing antibody ack2, by Thermo Fisher Scientific (2 mentions)
Recombinant scf, by Thermo Fisher Scientific (2 mentions)
Stem cell factor (scf), by Abcam (2 mentions)
Biotinylated anti mouse igg, by Vector Laboratories (2 mentions)
α smooth muscle actin α sma, by Merck Group (2 mentions)
9 protocols using fr180204
1
Anti-KIR2DL4 Antibody Signaling Pathway
2
Enrichment and Stimulation of Murine B Cells
3
Etoposide-Induced Kidney Cell Stress
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Human kidney proximal tubule cell line (HK-2) was purchased from American Type Culture Collection (ATCC, Manassas, VA, USA) and was maintained in RPMI1640 medium supplemented with 1% penicillin/streptomycin and 10% fetal bovine serum (Thermo Fisher Scientific Inc., Waltham, MA, USA). Cells were incubated at 37 °C in an atmosphere of 5% CO2 (Thermo Fisher Scientific Inc.). Cells (4 × 105) were seeded into a 6-cm dish (SPL Life Sciences., Pochun, South Korea) and incubated overnight at 37 °C in an atmosphere of 5% CO2. The medium was changed and etoposide (Sigma-Aldrich Co. LLC, St. Louis, MO, USA) was added as indicated. For MAPK inhibition experiments, cells were pre-treated for 1 hour with inhibitors of various signaling pathways: 10 μM SB203580 (p38MAPK inhibitor), 20 μM SP600125 (JNK inhibitor), 20 μM U0126 (inhibitors of MEK1 and MEK2, thus ERK) (Sigma-Aldrich Co. LLC), or 10 μM FR180204 (ERK 1/2 inhibitor) (Santa Cruz). For ROS inhibition experiments, cells were pre-treated with NAC or catalase (Sigma-Aldrich Co. LLC).
4
Enrichment and Stimulation of Murine B Cells
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Bone marrow cells were extracted from femurs and pelvises of euthanized mice, and single-cell suspensions were incubated for 3 min in ACK lysis buffer (0.15 M NH4Cl, 0.01 M KHCO3, and 0.1 mM EDTA [pH 7.2–7.4]) to remove erythrocytes. B cells were enriched by positive selection using anti-B220 mAbs coupled to magnetic beads (Miltenyi Biotec) and an AutoMACS (Miltenyi Biotec) according to the manufacturer’s instructions. B cell purity was consistently >95% based on B220 staining. Enriched B cells (or total bone marrow cells for experiments with ERK, AKT, and mTORC1 inhibitors) were cultured at 2 × 106 cells/ml at 37°C, 5% CO2, in complete IMDM (5% FBS, 1% GlutaMAX, 1% penicillin-streptomycin, 1% nonessential amino acids, 0.1 M 2-ME) for times indicated in the figure legends. The following inhibitors were added to the bone marrow B cell cultures: actinomycin D (0.1 μM; Sigma-Aldrich), afuresertib (5 μM; Sigma-Aldrich), bafilomycin (12 nM; Sigma-Aldrich), bortezomib (0.5 μM; Selleck Chemicals), cycloheximide (2 μM; Sigma-Aldrich), FR180204 (30 μM; Santa Cruz Biotechnology), and rapamycin (10 μM; EMD Millipore). The following Abs were added for B cell stimulation: anti-3–83Ig idiotypic Ab S23 (49 (link)) (produced in-house) at ≤10 μg/ml (as indicated in figures) and anti-IgM F(ab’)2 Abs (SouthernBiotech) at 5 μg/ml. DMSO and/or PBS diluents were added in control cultures.
5
Examining Placental Angiogenesis in Mice
6
Modulating STAT1 Acetylation Impacts Tau Biology
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The antibodies used in the present study are listed in the Supplementary Table S1 . TG-101348 (special JAK2 inhibitor, from MCE), JAK2 siRNA (sc-39099, from Santa Cruz Biotechnology), SP600125 (the inhibitor of JNK1, from Santa Cruz), and FR180204 (the inhibitor of ERK1, from Santa Cruz) were purchased. PcDNA3.0 vector-coded wild-type STAT1 (WT-STAT1) plasmid was the gift of Dr Xiao-Yuan Li (Institute of Biomedical Sciences, Academia Sinica, Taiwan), and PcDNA3.0 vector-coded wild-type STAT3 (WT-STAT3) plasmid was purchased from Shangdong Vigene Bioscience Biotechnology, co., LTD. EGFP-N1 vector coded mutant full-length human tau (P301L-hTau) plasmid, WT-STAT1 plasmid was mutated to pseudoacetylated STAT1 plasmids (single or double lysine sites (410, 413) mutated to glutamine, K410Q-, K413Q-, K410/413Q-STAT1) or unacetylated STAT1 plasmids (single or double lysine site (410, 413) mutated to arginine, K410R-, K413R-, K410/413R-STAT1) coded into RFP-N1 vector by Shanghai Baicheng Biotechnology, co., LTD.
7
Mouse Bone Marrow B Cell Isolation
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Bone marrow cells were extracted from femurs and pelvises of euthanized mice, and single-cell suspensions were incubated for 3 min in ACK lysis buffer (0.15 M NH4Cl, 0.01 M KHCO3, and 0.1 mM EDTA [pH 7.2–7.4]) to remove erythrocytes. B cells were enriched by positive selection using anti-B220 mAbs coupled to magnetic beads (Miltenyi Biotec) and an AutoMACS (Miltenyi Biotec) according to the manufacturer’s instructions. B cell purity was consistently >95% based on B220 staining. Enriched B cells (or total bone marrow cells for experiments with ERK, AKT, and mTORC1 inhibitors) were cultured at 2 × 106 cells/ml at 37° C,5% CO2, in complete IMDM (5% FBS, 1% GlutaMAX, 1% penicillin-streptomycin, 1% nonessential amino acids, 0.1 M 2-ME) for times indicated in the figure legends. The following inhibitors were added to the bone marrow B cell cultures: actinomycin D (0.1 μM; Sigma-Aldrich), afuresertib (5 μM; Sigma-Aldrich), bafilomycin (12 nM; Sigma-Aldrich), bortezomib (0.5 μM; Selleck Chemicals), cycloheximide (2 μM; Sigma-Aldrich), FR180204 (30 μM; Santa Cruz Biotechnology), and rapamycin (10 μM; EMD Millipore). The following Abs were added for B cell stimulation: anti-3-83Ig idiotypic Ab S23 (49 (link)) (produced in-house) at ≤10 μg/ml (as indicated in figures) and anti-IgM F(ab′)2 Abs (SouthernBiotech) at 5 μg/ml. DMSO and/or PBS diluents were added in control cultures.
8
Investigating Lipid Signaling Pathways
9
Examining Placental Angiogenesis in Mice
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Pregnant FVB/NJ mice, WBB6F1- Kit W− v/ + were obtained from Jackson Laboratories (Bal Harbor, ME). Primary antibodies utilized were: c-kit (1:50, DAKO Carpinteria, CA), SCF (1:500; Abcam, Cambridge, MA), α-Smooth Muscle Actin (α-sma: 1:500, Sigma-Aldrich; St. Louis, MO), von Willebrand factor (vWF: 1:200; DAKO), and Ki67 (1:100; Abcam). Secondary antibodies utilized were: Biotinylated anti-mouse IgG (1:200, Vector, CA), HRP Conjugated Donkey Anti-Goat IgG (1:2000, Jackson Immunoresearch, PA), Goat Anti-Mouse IgG Alkaline Phosphatase (1:100, Sigma-Aldrich), and Goat Anti-Rabbit IgG-Peroxidase (1:100, Sigma-Aldrich). Mouse c-kit neutralizing antibody (ACK2; 50μg/kg) and recombinant SCF were both obtained from EBioscience (San Diego, CA). FR180204 (a selective ERK1/2 antagonist) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA).
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