3-(4,5-Dimethylthiazol-yl)-2,5-diphenyltetrazolium bromide (MTT), propidium iodide (PI) and other chemicals were purchased from Sigma (St. Louis, Missouri, USA). The specific anti-SHP1 short interfering RNA (siRNA) and nonspecific control siRNA sequences were purchased from GenePharma (Shanghai, China). Commercial antibodies to the following antigens were as follows: SHP-1, EGFR, p-GSK3β (Ser9), Cyclin D1, and c-Myc (Epitomics, Burlingame, USA); p-EGFR, β-actin, GAPDH, histone H3, and EGFR (Santa Cruz Biotechnology, California, USA); h-Ras, p-Erk1/2, Erk1/2, β-catenin, Snail, E-cadherin, and N-cadherin (Cell Signaling Technology, Massachusetts, USA); k-Ras, GSK3β (Proteintech Group, Chicago, USA); and SHP-1 (Abcam, Cambridge, UK).
Shp 1
Manufactured by Abcam
Sourced in United States, United Kingdom
SHP-1 is a protein tyrosine phosphatase that plays a role in the regulation of cellular processes. It is expressed in various cell types and is involved in the negative regulation of cell signaling pathways. The core function of SHP-1 is to dephosphorylate specific target proteins, thereby modulating their activity and downstream effects.
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Lab products found in correlation
P stat3, by Cell Signaling Technology (6 mentions)
Stat3, by Cell Signaling Technology (6 mentions)
Stat3, by Abcam (5 mentions)
P stat3, by Abcam (5 mentions)
Survivin, by Cell Signaling Technology (5 mentions)
Ptp1b, by Abcam (4 mentions)
Gapdh, by Cell Signaling Technology (4 mentions)
Cyclin d1, by Abcam (4 mentions)
Mcl 1, by Abcam (4 mentions)
Mcl 1, by Cell Signaling Technology (4 mentions)
β actin, by Abcam (4 mentions)
Poly adp ribose polymerase parp, by Cell Signaling Technology (3 mentions)
Polyvinylidene difluoride membrane, by Merck Group (3 mentions)
Cleaved caspase 3, by Cell Signaling Technology (3 mentions)
Shp 2, by Abcam (3 mentions)
Bcl 2, by Abcam (2 mentions)
Caspase 3, by Cell Signaling Technology (2 mentions)
Caspase 9, by Cell Signaling Technology (2 mentions)
Cyclin d1, by Santa Cruz Biotechnology (2 mentions)
P jak1, by Cell Signaling Technology (2 mentions)
25 protocols using shp 1
1
MTT Assay and Signaling Pathway Analysis
2
Western Blot Analysis of Protein Expression
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Total protein was extracted by radio-immunoprecipitation assay (RIPA) buffer containing phosphatase and protease inhibitors (Beyotime, Shanghai, China). Equal amounts of protein from each sample were separated by 10% or 12% sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE), and then electro-transferred onto nitrocellulose membranes (Millipore, Bredford, MA, USA). Following blocking with 5% skim milk in Tris-buffered saline Tween-20 (TBST) for 30 min at room temperature, the membranes were probed with the following primary antibodies: TRIM52 antibody from San Cruz (Santa Cruz, CA, USA), Bcl-2, Bax, p-STAT3, STAT3, PTP1B, TCPTP, SHP1, SHP2 and ubiquitin (Ub) antibodies from Abcam (Cambridge, MA, USA), and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) antibody from Cell Signaling Technology (Danvers, MA, USA) according to the manufacturers’ protocols. After being rinsed by TBST, the membranes were incubated with incubated with peroxidase labeled secondary antibody at room temperature for 1 h. Signal was detected with enhanced chemiluminescence substrate (ECL, BioRad, Richmond, CA, USA).
3
Nintedanib Modulates Signaling Pathways in Triple-Negative Breast Cancer
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MDA-MB-231, MDA-MB-468 and HCC-1395 cell lines were obtained from the ATCC (American Type Culture Collection, Rockville, MD, USA) and maintained in Dulbecco’s Modified Eagle Medium (DMEM, Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS, HyClone, Thermo Scientific, Waltham, MA, USA). Cell lysates treated with nintedanib were prepared and analyzed by western blot as previously reported.18 (link) Antibodies targeting p-JAK2 (Tyr1007/1008), JAK2, p-SRC (Tyr416), SRC, p-STAT3 (Tyr705), STAT3, survivin, poly (ADP-ribose) polymerase (PARP) and cleaved caspase 3 were purchased from Cell Signaling (Danvers, MA, USA). SHP-1, cyclin D1 and Mcl-1 antibodies were purchased from Abcam (Cambridge, MA, USA).
4
Huh7 and PLC5 Cell Line Maintenance
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The Huh7 HCC cell line was obtained from the Health Science Research Resources Bank (HSRRB; Osaka, Japan; JCRB0403). The PLC/PRF/5 (PLC5) cell lines were obtained from American Type Culture Collection (ATCC). All cells obtained from HSRRB or ATCC were immediately expanded and frozen down such that all cell lines could be restarted every 3 months from a frozen vial of the same batch of cells. No further authentication was conducted in our laboratory. Cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% FBS, 100 units/mL penicillin G, 100 mg/mL streptomycin sulfate, and 25 mg/mL amphotericin B in a humidified incubator at 37 °C in an atmosphere of 5% CO2 in air. Results from all in vitro studies were confirmed in at least three independent experiments to verify results. Antibodies for immunoblotting such as p-STAT3(Tyr705), STAT3, survivin, caspase-3 and caspase-9 were from Cell Signaling (Danvers, MA). SHP-1, cyclin D1 and Mcl-1 antibodies were purchased form Abcam (Cambrige, MA). RFX-1 antibody was purchased from Novus Biologicals (Colorado, USA).
5
Western Blot Analysis of Cell Lysates and Exosomes
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Western blotting of cell lysates and exosomes were performed according to standard protocols. Proteins blotted to Hy-bond nylon members (Amersham) were detected by antibodies against SHP-1 (Abcam), PTP1B (Abcam), TCPTP (Abcam), GP63 (Robert McMaster, University of British Columbia, Canada), LACK (Jean-Claude Antoine Institute Pasteur, France, post-humous), HSP83 (Greg Matlashewski, McGill University, Canada), and tubulin (Abcam). Anti-mouse or anti-rabbit antibodies conjugated to horse-radish peroxidise (HRP) (Amersham) were used as secondary antibodies. Membranes were then visualized by ECL Western blotting detection system (Amersham).
6
Protein Expression Analysis by Western Blot
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Whole-cell lysates were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Proteins were transferred onto a polyvinylidene difluoride membrane (Millipore, Billerica, MA) and incubated with the primary antibody, and then incubated with horseradish peroxidase–conjugated secondary antibodies. Specific proteins were detected using enhanced chemiluminescence reagent. The primary antibodies used for Western blotting, including cyclin D1 and PARP, were purchased from Santa Cruz Biotechnology (San Diego, CA); p-STAT3, STAT3, Mcl-1 and survivin were purchased from Cell Signaling (Danvers, MA); SHP-1 and β-actin were purchased from Abcam (Cambridge, MA). For quantification of protein levels on chemiluminescent Western blots, the densitometry Image J software was employed.
7
Synthesis and Evaluation of the SHP-1 Inhibitor SC-60
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SC‐60 was synthesized by Hinova Pharmaceuticals Inc. (Chengdu, China), the MW of SC‐60 is 473, and its structure and solubility are described in Fig. S1 . For in vitro studies, SC‐60 at various concentrations was dissolved in dimethyl sulfoxide (DMSO) and added to cells in Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad, CA, USA). The final DMSO concentration was 0.1% after addition to the medium. For in vivo studies, SC‐60 was dissolved in 50% (v/v) propylene glycol and 50% (v/v) Solutol® HS‐15. The SHP‐1 inhibitor PTP inhibitor III (CAS 29936‐81‐0) was purchased from Cayman Chemical (Ann Arbor, MI, USA). Plasmids of human wild‐type STAT3 were encoded by pCMV6 vector with myc‐tag. The mutant SHP‐1 constructs (DN1 and D61A) have been generated to mimic the open‐form structure of SHP‐1 as previously described (Tai et al., 2014b ). Antibodies for immunoblotting such as p‐VEGFR2, VEGFR2, p‐PDGFRβ, PDGFβ, p‐JAK1, JAK1, p‐JAK2, JAK2, p‐SRC, SRC, p‐STAT3, STAT3, and survivin were from Cell Signaling (Danvers, MA, USA). SHP‐1, cyclin D1, and Mcl‐1 antibodies were purchased from Abcam (Cambridge, MA, USA). Other antibodies such as poly (ADP‐ribose) polymerase (PARP) and cleaved caspase 3 were obtained from Cell Signaling Technology.
8
Western Blot Analysis of Cellular Proteins
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Whole-cell lysates were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were transferred onto a polyvinylidene difluoride membrane (Millipore, Billerica, MA, USA) and incubated with the primary antibody, and then incubated with horseradish peroxidase-conjugated secondary antibodies. Specific proteins were detected using enhanced chemiluminescence (ECL) reagent. The primary antibodies used for western blotting, including cyclin D1 and PARP were purchased from Santa Cruz Biotechnology (San Diego, CA); p-STAT3, STAT3, Mcl-1 and Caspase-9 were purchased from Cell Signaling (Danvers, MA); SHP-1 and β-actin were purchased from Abcam (Cambrige, MA).
9
STAT3, SMAD2, and JAK1 Signaling Antibodies
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Antibodies against STAT3 (#9139; 1/1,000), pY705-STAT3 (#9145; 1/1,000), ac-K685 STAT3 (#2523; 1/1,000), SMAD2 (#3122; 1/1,000), pSer465/467-SMAD2 (#3108; 1/1,000), pY1022/1023-JAK1 (#3331; 1/200), JAK1 (#3332; 1/500), MLC2 (#3672; 1/500) and pThr18/19-MLC2 (#3674; 1/500) were purchased from Cell Signalling (Cell SignalingTechnology, Beverly MA), α-tubulin from sigma (T4026, Sigma, Saint Louis, MO; 1/5,000); SHP-1 (#sc-7289, 1/ 500 for western blot analysis), DNMT1 (#sc-20701; 1/1,000), DNMT3b (#sc-130740; 1/1,000) from Santa Cruz Biotechnology (Santa Cruz Biotechnology, Santa Cruz, CA) and SHP-1 (#ab2020; 1/200 for immunohistochemical staining) from Abcam (#ab2020, Abcam, Cambridge, UK) from Bethyl Laboratories (Bethyl Laboratories, Montgomery, TX, USA).
10
Western Blot Analysis of Key Signaling Proteins
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Cells were lysed in RIPA buffer (Beyotime, China). The protein was separated in 10% SDS-PAGE gel and transferred to nitrocellulose membranes. The 5% non-fat milk was applied to the membrane to block non-specific antigens. After incubation with primary and secondary antibodies (Beyotime Biotech.), the protein content was detected using an ECL kit (Pierce, USA). Primary antibodies used in this study were as follows: UBE2D3 (Abcam, Ab176568); STAT3 (Abcam, Ab68153); p-STAT3 (Abcam, Ab76315); HK2 (Abcam, Ab209847); PFKL (Abcam, Ab181064); PTP1B (Abcam, Ab244207); SHP-1 (Abcam, Ab32559); SHP-2 (Abcam, Ab32159); SOCS1 (Abcam, Ab62584); SOCS3 (Abcam, Ab16030); Ubiquitin (Abcam, Ab7780); β-actin (Abcam, Ab8227).
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