A RediPlate 96 EnzChek Tyrosine Phosphatase Assay Kit (R-22067) was used for SHP-1 activity assay (Molecular Probes, Carlsbad, CA). The method was as described previously52 (link).
Shp 1 activity assay
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The SHP-1 activity assay is a laboratory instrument designed to measure the enzymatic activity of the SHP-1 (Src Homology 2 domain-containing Protein Tyrosine Phosphatase-1) protein. SHP-1 is a phosphatase enzyme that plays a crucial role in regulating cellular signaling pathways. The assay provides a quantitative assessment of SHP-1 activity, which can be useful in various research applications involving cellular signaling and immune system regulation.
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10 protocols using shp 1 activity assay
1
SHP-1 Phosphatase Activity Assay
2
Sorafenib, Brivanib, and Sunitinib Effects on SHP-1 Activity
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After treatment with sorafenib, brivanib, or sunitinib, HCC protein extract was incubated with anti-SHP-1 antibody in immunoprecipitation buffer (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1% NP-40, and 1% sodium deoxycholate) overnight. Protein G-Sepharose 4 Fast flow (GE Healthcare Bio-Science, NJ) was added to each sample, followed by incubation for 3 h at 4°C with rotation. A RediPlate 96 EnzChek Tyrosine Phosphatase Assay Kit (R-22067) was used for SHP-1 activity assay (Molecular Probes, Invitrogen, CA).
3
SHP-1 Phosphatase Activity Assay
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After treatment with regorafenib, cell protein extracts were incubated with anti-SHP-1 antibody in immunoprecipitation buffer (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1mM EDTA, 1% NP-40, and 1% sodium deoxycholate) overnight. Protein G-Sepharose 4 Fast flow (GE Healthcare Bio-Science, NJ) was added to each sample, followed by incubation for 3 h at 4°C with rotation. A RediPlate 96 EnzChekR Tyrosine Phosphatase Assay Kit (R-22067) was used for SHP-1 activity assay (Molecular Probes, Invitrogen, CA).
4
Quantifying SHP-1 Phosphatase Activity
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A RediPlate 96 EnzChek® Tyrosine Phosphatase Assay Kit (R-22067) was used for SHP-1 activity assay (Molecular Probes, Invitrogen, CA). To detect the phosphatase activity of endogenous SHP-1, SHP-1 protein in Huh-7 cell lysates was incubated with anti-SHP-1 antibody at 4 °C overnight and precipitated with Protein G-Agarose beads (Roche). The beads were washed with immunoprecipitation buffer (20 mM Tris-HCl (pH 8.0), 50 mM NaCl, 0.5% Triton-X-100, and 10% glycerol) and placed into RediPlate wells. SHP-1 activity was measured at 360/40 and 460/40 nm. The amount of SHP-1 protein used for the phosphatase assay were evaluated by western blotting. For in vitro phosphatase assay, 4 nM recombinant His-SHP-1 or His-SHP-1Δ517–597 protein and 4 nM HNF1A-AS1 were added into RediPlate wells and incubated for 30 min at RT before reading fluorescence.
5
Measuring SHP-1 Phosphatase Activity
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After treatment with SC-43, cell protein extracts were incubated with anti–SHP-1 antibody in immunoprecipitation buffer [20 mM Tris-Hcl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1% NP-40, and 1% sodium deoxycholate] overnight. Protein G-Sepharose 4 Fast Flow (GE Healthcare Bio-Science, NJ) was added to each sample, followed by incubation for 3 hours at 4°C with rotation. A Rediplate 96 Enzchekr Tyrosine Phosphatase Assay Kit (R-22067) was used for SHP-1 activity assay (Molecular Probes, Invitrogen, CA). For the examination of the SHP-1 activity in the SHP-1–containing immunoprecipitation of cellular extract, 1 mg of whole-cell lysates was incubated with anti–SHP-1 antibody in immunoprecipitation buffer overnight. Then, samples were incubated with Protein G-Sepharose 4 Fast Flow for 3 hours at 4°C with rotation and finally treated with SC-43 for 2 hours at 4°C. The SHP-1 activity in SHP-1–containing immunoprecipitation of cellular extract following SC-43 treatment was determined by using a Rediplate 96 Enzchekr Tyrosine Phosphatase Assay Kit.
6
SHP-1 Phosphatase Activity Assay
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The Rediplate 96 EnzChek®Tyrosine Phosphatase Assay Kit (R-22067) was used for SHP-1 activity assay (Molecular Probes, Carlsbad, CA). The SHP-1 phosphatase activity was measured as described before [30 (link)].
7
SHP-1 Phosphatase Activity Assay
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A RediPlate 96 EnzChek Tyrosine Phosphatase Assay Kit (R-22067) was used for the SHP-1 activity assay (Molecular Probes, Carlsbad, CA). The method was as described previously [16] (link).
8
SHP-1 Phosphatase Activity Assay
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After treatment of SC-43, the cellular protein extracts were incubated with anti-SHP-1 antibody in immunoprecipitation buffer (20 mM of Tris-HCl [pH 7.5], 150 mM of NaCl, 1 mM of ethylenediaminetetraacetic acid, 1% NP-40, and 1% sodium deoxycholate) overnight. Protein G-Sepharose 4 Fast flow (GE Healthcare Bio-Science, NJ, USA) was added to each sample, followed by incubation for 3 h at 4°C with rotation. A RediPlate 96 EnzChekR Tyrosine Phosphatase Assay Kit (R-22067) was used for SHP-1 activity assay (Molecular Probes, Invitrogen, CA, USA).
9
SHP-1 Phosphatase Activity Assay
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A RediPlate 96 EnzChek Tyrosine Phosphatase Assay Kit (R‐22067) was used for SHP‐1 activity assay (Molecular Probes, Carlsbad, CA, USA). The method was as described previously (Liu et al., 2013 ). Briefly, the protein extracts of breast cancer cells were incubated with anti‐SHP‐1 antibody in immunoprecipitation buffer overnight. Then, the Protein G‐Sepharose 4 Fast Flow (GE Healthcare, Piscataway, NJ, USA) was added to each sample followed by incubation for three hours at 4 °C with rotation and then assayed for phosphatase activity.
10
SHP-1 Phosphatase Activity Assay
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The cellular protein extracts were incubated with anti-SHP-1 antibody in immunoprecipitation buffer overnight. Protein G-Sepharose 4 Fast flow (GE Healthcare Bio-Science, NJ) was added to each sample, followed by incubation for 3 h at 4°C with rotation. A RediPlate 96 EnzChekR Tyrosine Phosphatase Assay Kit (R-22067) was used for SHP-1 activity assay (Molecular Probes, Invitrogen, CA).
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