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P shp1

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P-SHP1 is a recombinant protein that is phosphorylated on tyrosine 536. It is a useful tool for studying the regulation and function of the SHP1 (PTPN6) protein, which is a tyrosine phosphatase involved in cellular signaling pathways.

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Lab products found in correlation

β actin, by Cell Signaling Technology (2 mentions) Fluorescein isothiocyanate (fitc), by Thermo Fisher Scientific (1 mentions) P stat1, by Cell Signaling Technology (1 mentions) P nf κb, by Cell Signaling Technology (1 mentions) α tubulin, by Cell Signaling Technology (1 mentions) Gapdh, by Cell Signaling Technology (1 mentions) Zap70, by Cell Signaling Technology (1 mentions) P p44 42 mapk t202 y204, by Cell Signaling Technology (1 mentions) Ifn γ 4s b3, by Thermo Fisher Scientific (1 mentions) Pzap70 py319, by Cell Signaling Technology (1 mentions) P akt, by Cell Signaling Technology (1 mentions) P erk, by BD (1 mentions) Perm wash solution, by Thermo Fisher Scientific (1 mentions) Pplcγ2, by BD (1 mentions) P cd79a, by Cell Signaling Technology (1 mentions) P pi3k p85, by Cell Signaling Technology (1 mentions) Pvdf membrane, by Merck Group (1 mentions) Ripa lysate, by Beyotime (1 mentions) Pgc 1α, by Cell Signaling Technology (1 mentions) Ecl luminescent substrate, by Merck Group (1 mentions)

4 protocols using p shp1

1

MDSC Stimulation and Signaling Pathway Analysis

Cited 1 time
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MDSC were stimulated with GA for 2 hr, followed by treatment with vehicle, LPS (100 ng/ml), or IFNγ (20 ng/ml) for an additional 30 min. Total cell lysates were prepared for immunoblotting. p-NF-κB, p-p38, p-STAT1, p-SHP1, β-actin, Tubulin (Cell Signaling Technology), FITC (Thermo Fisher Scientific) and PIR-B (R&D systems) detection antibodies were used according to manufacturer’s recommendations. For immunoprecipitation, total cell lysates from CD115+ MDSC were incubated with PBS vehicle control or GA-FITC, followed by anti-FITC antibody detection and protein G bead pulldown according to manufacturer’s recommendations (Thermo Fisher Scientific). The precipitates and total lysates were analyzed by immunoblot as previously described (22 (link)).
van der Touw W., Kang K., Luan Y., Ma G., Mai S., Qin L., Bian G., Zhang R., Mungamuri S.K., Hu H.M., Zhang C.C., Aaronson S.A., Feldmann M., Yang W.C., Chen S.H, & Pan P.Y. (2018). Glatiramer acetate enhances myeloid-derived suppressor cell function via recognition of paired immunoglobulin-like receptor-B. Journal of immunology (Baltimore, Md. : 1950), 201(6), 1727-1734.
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2

Characterization of T Cell Signaling Pathways

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Antibodies against HLA-A2 (BB7.2), HLA-ABC (W6/32), CD8a (RPA-T8), TNF (MAb11), and IFN-γ (4 S.B3) were purchased from eBioscience. Anti-CD3 (SK7), CD107a (H4A3), CD247-Alexa Fluor 488 (pY142) (K25-407.69), pCD3ζ (pY142) (K25-407.69), Erk1 (Cat# 610031), and PLC-γ1 (Cat# 610027) were from Becton Dickinson. Anti-pLAT (Tyr191) (Cat# 3584S), P-p44/42 MAPK (T202/Y204) (Cat# 4370L), pSrc family (pY416) (Cat# 6943T), non-pSrc family (pY416) (Cat# 2102S), pSHP-1 (Cat# 8849S), pZap70 (pY319) (Cat# 2717S), Zap70 (Cat# 2709), pPLC-γ1 (pY783) (Cat# 2821S), α-tubulin (Cat# 3873S), GAPDH (Cat# 2118S) antibodies were from Cell Signaling Technologies. Anti-LAT (11B.12) and β-2-microglobulin (2M2) antibodies were from Biolegend. Mouse anti-human HLA-A2-E183-91 and mouse antihuman HLA-A2-C18-27 primary antibodies were produced as described18 (link). F(ab’)2-goat anti-rabbit IgG (H + L) cross-adsorbed secondary antibody, Alexa Fluor 488 (Cat# A-11070), and goat anti-mouse IgG (H + L) cross-adsorbed secondary antibody, Alexa Fluor 647 (Cat# A-21237) were purchased from Thermo Fisher Scientific.
Zhao X., Sankaran S., Yap J., Too C.T., Ho Z.Z., Dolton G., Legut M., Ren E.C., Sewell A.K., Bertoletti A., MacAry P.A., Brzostek J, & Gascoigne N.R. (2018). Nonstimulatory peptide–MHC enhances human T-cell antigen-specific responses by amplifying proximal TCR signaling. Nature Communications, 9, 2716.
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3

Phosphoflow Analysis of B Cell Signaling

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For phosphoflow experiments, 0.5 × 106 cells were cultured in RPMI‐2% FCS at 37°C for 1 min for pPI3K p85 (Cell Signaling Technologies), 3 min for pSHP‐1 (Cell Signaling Technologies), 5 min for pSrc (Abcam), pLyn (Abwiz Bio, San Diego, CA, USA), pCD79a (Cell Signaling Technologies), pSyk, pSLP‐65, pPLCγ2, and pErk (all BD Biosciences) or for 3 h for pS6 and pAkt (both Cell Signaling Technologies) with 25 μg/mL anti‐IgM or antigen‐binding F(ab’)2‐Igκ fragments (anti‐IgK; Jackson Immunoresearch). After fixation with the eBioscience FoxP3 staining kit Fix/Perm solution, cells were washed twice with the accompanying Perm/Wash solution (Invitrogen). Next, cells were incubated with varying combinations of monoclonal antibodies (Supporting information Table S1) and stained according to previously described procedures [31 (link)]. After staining for the phosphoproteins, samples were measured in MACS buffer on an LSR‐II (BD Biosciences) and analyzed using FlowJo v10 (BD Biosciences).
Rip J., de Bruijn M.J., Neys S.F., Singh S.P., Willar J., van Hulst J.A., Hendriks R.W, & Corneth O.B. (2021). Bruton's tyrosine kinase inhibition induces rewiring of proximal and distal B‐cell receptor signaling in mice. European Journal of Immunology, 51(9), 2251-2265.
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4

Western Blot Analysis of Liver Proteins

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The liver tissue and cell proteins were extracted with RIPA lysate (Beyotime Biotechnology), and the concentration was determined using the BCA method. The protein was separated by 10% acrylamide gel electrophoresis and transferred to the PVDF membrane (Millipore, Darmstadt, and Germany). TBST solution containing 5% skim milk (BD, Maryland, United States) was used to block the membrane for 1 h, followed by incubation with primary antibodies at 4°C overnight. Antibodies against P-JNK, JNK, P-Src, Src, P-SHP-1, SHP-1, Peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), and β-actin were purchased from Cell Signaling (Massachusetts, United States). Antibodies against Sab and Cleaved PARP were obtained from Proteintech (Wuhan, China) and ABclonal (Wuhan, China) respectively. The membrane was then incubated with the secondary antibodies at room temperature for 1 h and subsequently incubated with ECL luminescent substrate (Millipore, Billerica, United States). The signals were acquired using Tanon-5200 chemiluminescence image analysis system (Shanghai, China).
Jiang Y., Xu J., Huang P., Yang L., Liu Y., Li Y., Wang J., Song H, & Zheng P. (2022). Scoparone Improves Nonalcoholic Steatohepatitis Through Alleviating JNK/Sab Signaling Pathway-Mediated Mitochondrial Dysfunction. Frontiers in Pharmacology, 13, 863756.
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