Phosphatases

Manufactured by Merck Group

Phosphatases are enzymes that catalyze the hydrolysis of phosphate groups from various substrates, such as proteins, nucleic acids, and other molecules. They play a crucial role in regulating cellular processes by modulating the phosphorylation state of their targets.

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Cocktail of proteases and phosphatases inhibitors Protease and phosphatases inhibitor cocktails Proteases and phosphatases inhibitors Phosphatases inhibitors Proteases and phosphatases inhibitors cocktails Anti-proteases and anti-phosphatases Alkaline Phosphatase Blue Membrane Substrate Solution Anti-polyhistidine–alkaline phosphatase antibody Monoclonal anti-Myc and anti-His alkaline phosphatase (AP) conjugated antibodies Leukocyte Acid Phosphatase (TRAP) kit

7 protocols using phosphatases

Extraction and Quantification of Soluble and Membrane Plant Proteins

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Frozen plant material was suspended in 2ml extraction buffer containing 250mM sucrose, 20mM Tris-HCl (pH 7.5), 10mM EDTA, 1mM 1,4-dithiothreitol (DTT), and inhibitor cocktail for proteases (Sigma) and phosphatases (Sigma). Cell debris was removed by centrifugation at 8000 g for 10min at 4 °C. Supernatants were transferred to clean tubes and centrifuged at 120 000 g for 1h at 4 °C. The final supernatants were used for soluble protein extraction, as described previously (Wang et al., 2009 (link)). The pellets (membrane-associated proteins) were re-precipitated with acetone overnight at –20 °C. The precipitated membrane proteins were rinsed three times with ice-cold acetone containing 13mM DTT and subsequently lyophilized. Finally, both protein pellets were resuspended separately by adding 8M urea, 4% CHAPS, 65mM DTT, and 40mM Tris (pH 7.5). The protein concentration was determined according to Peterson (1977) (link) using BSA as a standard. The supernatants were stored in aliquots at –80 °C or directly digested with trypsin.

Wang X., Qi M., Li J., Ji Z., Hu Y., Bao F., Mahalingam R, & He Y. (2014). The phosphoproteome in regenerating protoplasts from Physcomitrella patens protonemata shows changes paralleling postembryonic development in higher plants. Journal of Experimental Botany, 65(8), 2093-2106.

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Western Blot Analysis of Protein Expression

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Samples taken from cells or lung tissues from same batch of mice infected as above were lysed with RIPA buffer (30 mM Tris-HCl, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 10% glycerol, and complete cocktail (Roche) and phosphatases (Sigma). Lysates were centrifuged at 14000×g for 15 min, the supernatants were collected and the concentration was quantitated. The Samples were boiled for 10 min, and equal amount was applied to 12% SDS-polyacrylamide minigels and electrophoresed. The proteins in the gel were then transferred to nitrocellulose filter membranes (Thermo, Rockford, IL). Horseradish peroxidase (HRP)-linked secondary antibody (Rockland, Gilbertsville, PA) and X-ray film (Kodak) were used for exposure (20 (link), 21 (link)). Mouse polyclonal antibody anti-LC3, Beclin1, Bcl-2, Bax, Cytochrome C, rabbit polyclonal antibody anti-IFN-γ, pJAK2, JAK2, STAT1α, IRF1, SHP2, β-actin and goat polyclonal antibody anti-Atg5-Atg12 and pSTAT1α were bought from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit monoclonal antibody anti-cleaved-PARP and cleaved-caspase3 were bought from Cell Signaling Technology (Danvers, MA).

Li X., Ye Y., Zhou X., Zhao K., Huang C, & Wu M. (2014). Atg7 enhances host defense against infection via down-regulation of superoxide but up-regulation of nitric oxide. Journal of immunology (Baltimore, Md. : 1950), 194(3), 1112-1121.

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Western Blot Analysis of Protein Expression

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Samples taken from either cells or tissues of mice after experimental treatment were lysed with RIPA buffer (30 mM Tris-HCl, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 10% glycerol, and complete cocktail (Life technologies, Grand Island, NY) and phosphatases (Sigma, St. Louis, MO). Lysates were centrifuged at 14000×g for 15 min, the supernatants were collected and the concentration was quantitated. The samples were boiled for 10 min, and equal amount was applied to 12% SDS-polyacrylamide minigels and electrophoresed. The proteins in the gel were then transferred to nitrocellulose filter membranes (Thermofisher, Rockford, IL). Horseradish peroxidase (HRP)-linked secondary antibody (Rockland, Gilbertsville, PA) and X-ray film (Kodak) were used for exposure ^{47 (link),48 (link)}. Rabbit polyclonal IgG antibody anti-GAPDH, c-Myb, MyD88, TLR4, PKA, pPKA (Thr 198), CREB-1, pCREB-1 (Ser 133), pp65 (Ser 536), TGF-β1, goat polyclonal IgG antibody anti-IL-4, and mouse monoclonal IgG antibody anti-p65 were bought from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit polyclonal IgG antibody anti-TRIF were bought form Abcam (Cambridge, MA).

Li X., He S., Li R., Zhou X., Zhang S., Yu M., Ye Y., Wang Y., Huang C, & Wu M. (2016). Pseudomonas aeruginosa infection augments inflammation through miR-301b repression of c-Myb-mediated immune activation and infiltration. Nature microbiology, 1(10), 16132.

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Western Blot Analysis of Protein Expression

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Western Blot Analysis of STAT5 Signaling

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Ice-cold RIPA buffer (15 mM NaCl, 50 mM Tris HCL PH 7.4, 1 mM EDTA, 0.5 mM EGTA, 1% Triton, 0.1% SDS, 0.1% sodium deoxycholate) in the presence of proteases (Roche) and phosphatases (Sigma) inhibitors cocktails was used to cell lysis. After 30 min of ice incubation, the lysates were cleared via centrifugation at 16,000 g for 15 min at 4 °C, and the protein concentration was determined using the BioRad Dc protein assay as recommended by the manufacturer (Bio-Rad, Marnes-La-Coquette, France). Proteins were mixed with 5X Laemmli loading buffer and incubated 10 min at 95 °C. Fifty μg of proteins were migrated in a 10% SDS-PAGE and transferred in a nitrocellulose membrane. The membranes were probed overnight at 4 °C with primary specific antibodies (STAT5, pSTAT5 (Cell Signaling, Danvers, MA, USA), HSC70 (Santa Cruz, Santa Cruz, CA, USA) and FATP2 (Abcam, Cambridge, UK)) after 45 min of non-specific binding sites blocking with TBS 0.1% Tween 5% nonfat milk (pSTAT5) or bovine serum albumin (STAT5, FATP2 and HSC70). The membranes were washed and incubated for 1 h at room temperature with horseradish peroxidase-conjugated secondary antibody (Jackson Immunoresearch Laboratories). Immunoblot was revealed using Clarity Western ECL Substrate kit and ChemiDoc imaging system (Bio-Rad).

Mabrouk N., Racoeur C., Shan J., Massot A., Ghione S., Privat M., Dondaine L., Ballot E., Truntzer C., Boidot R., Hermetet F., Derangère V., Bruchard M., Végran F., Chouchane L., Ghiringhelli F., Bettaieb A, & Paul C. (2023). GTN Enhances Antitumor Effects of Doxorubicin in TNBC by Targeting the Immunosuppressive Activity of PMN-MDSC. Cancers, 15(12), 3129.

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Western Blot Analysis of HO-1 Induction in MDM

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HO-1 induction in MDM was determined by Western blot (Enzo Life Sciences, Farmingdale, NY). Briefly, total cell protein extracts were prepared in modified radioimmunoprecipitation assay buffer (50 mM Tris-HCl, 1% NP-40, 0.25% deoxycholic acid, 150 mM NaCl, 1 mM EGTA, 1 mM sodium orthovanadate, and 1 mM sodium fluoride) containing inhibitors of proteases (Roche Applied Science) and phosphatases (Sigma). After quantification using the Pierce BCA protein assay, total protein (2.5 to 5 μg per lane) was resolved on a 12% Tris-glycine sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE) gel. After transfer to a polyvinylidene difluoride membrane (Millipore), HO-1 and actin were detected using 1:5000 dilutions of primary rabbit polyclonal antibodies, followed by a horseradish peroxidase-conjugated anti-rabbit IgG. Protein bands were visualized using the ECL^® (enhanced chemiluminescence) detection system (GE Healthcare). Western blots were performed by Kendrick Labs, Inc. (Madison, WI).

Huang H., Zhou Z.H., Adhikari R., Yamada K.M, & Dhawan S. (2016). Defective iron homeostasis in human immunodeficiency virus type-1 latency. Current trends in immunology, 17, 125-131.

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Western Blot Protein Analysis Protocol

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Whole cell proteins were extracted in lysis buffer (150mM NaCl, 25mM Tris pH7.5, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) supplemented with inhibitors of proteases (Roche) and phosphatases (Sigma). Equal amounts of proteins were resolved on SDS-PAGE 4-12% (Bio-Rad) and transferred onto PVDF membranes. Membranes were blocked using 5% non-fat dry milk in TBS-Tween 0.1% (TBST) for 1h at room and probed with primary antibodies overnight at 4°C. The list of antibodies is available in Supplementary Table 1. After 4 washes in TBST, membranes were incubated 1h with HRP-conjugated secondary antibodies at 1:5,000 (Bio-Rad). After 4 more washes, immunoblots were developed by enhanced chemiluminescence. When required, PVDF membranes were stripped in 62.5mM Tris HCl, pH6.8, 2% SDS and 0.8% β-mercaptoethanol.

Brun C., Sincennes M., Lin A.Y., Hall D.T., Jarassier W., Feige P., Ritso M., Grand F.L., & Rudnicki M.A. (2020). GLI3 Processing by the Primary Cilium Regulates Muscle Stem Cell Entry into G_Alert.

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