Phosphatase inhibitor cocktail

Manufactured by Thermo Fisher Scientific

Sourced in United States, Switzerland, Germany, Japan, United Kingdom, China

Phosphatase inhibitor cocktail is a liquid solution designed to inhibit the activity of phosphatases, a class of enzymes that remove phosphate groups from proteins. This product can be used to preserve the phosphorylation state of proteins in biological samples, which is important for various analytical and research applications.

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Cocktails of proteases and phosphatases inhibitors Proteases and Phosphatases Inhibitor Cocktail HALT Protease and Phosphatase Inhibitor cocktails without EDTA Protease/phosphatase inhibitors cocktail Cocktail of protease and phosphatase inhibitors Cocktail of phosphatase inhibitors Protease and phosphatase inhibitors cocktail Halt protease and phosphatase inhibitors cocktail Halt™ Protease & Phosphatase Inhibitors Cocktail Cocktail of protease/phosphatase inhibitors

383 protocols using phosphatase inhibitor cocktail

Stage-specific Parasite Extraction

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For the stage-specific pull-down analysis parasites were harvested at ring stage (approx. 12HPI), trophozoites (approx. 24HPI) and schizonts (approx. 36HPI) Parasites were liberated, and red blood cells lysed using 10 volumes of 0.1% (w/v) saponin (Sigma) in ice-cold 1 x PBS (1^st Base) for 5 minutes. Parasite pellet was washed three times with 1 x PBS and the pellet was lysed (for most of assays except Pull-downs) with RIPA buffer (150 mM sodium chloride (Merck); 1% Triton X-100 (BioRad); 0.5% sodium deoxycholate (Sigma); 0.1% sodium dodecyl sulfate (BioRad); and 50mM Tris (BioRad) pH8.0) supplemented with 1% (v/v) protease inhibitor cocktail (Nacalai Tesque) and 1% (v/v) phosphatase inhibitor cocktail (Thermo Fisher). Samples were incubated for 30 min at 4°C with gentle agitation. For native pull-down assays RIPA buffer was substituted with Pierce IP Lysis Buffer (Thermo Fisher) with 1% (v/v) protease inhibitor cocktail (Nacalai Tesque) and 1% (v/v) phosphatase inhibitor cocktail (Thermo Fisher). Protein lysate debris was removed by centrifugation (15 min at 4°C) and protein concentration in the supernatant was quantified using Pierce BCA Protein Assay Kit (Thermo Scientific). Samples were frozen with liquid nitrogen and stored at -80°C.

Kucharski M., Wirjanata G., Nayak S., Boentoro J., Dziekan J.M., Assisi C., van der Pluijm R.W., Miotto O., Mok S., Dondorp A.M, & Bozdech Z. (2023). Short tandem repeat polymorphism in the promoter region of cyclophilin 19B drives its transcriptional upregulation and contributes to drug resistance in the malaria parasite Plasmodium falciparum. PLOS Pathogens, 19(1), e1011118.

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Immunoblot Analysis of Protein Expression

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The immunoblot analysis was performed as described previously.^{15 (link)} Briefly, cells were lysed with RIPA buffer (20–188, Millipore) supplemented with protease inhibitor cocktail and phosphatase inhibitor cocktail (Thermo Fisher Scientific). For the separation of nuclear and cytoplasmic proteins, cells were firstly lysed with cytoplasmic lysis buffer (Tris 10 mM, NaCl 10 mM, MgCl2 3 mM, Nonidet P-40 0.1%) supplemented with protease inhibitor cocktail and phosphatase inhibitor cocktail (Thermo Fisher Scientific) for 3 min, and the supernatant was collected for the detection of cytoplasmic proteins. After three washes with cytoplasmic lysis buffer, the nuclei were lysed with RIPA buffer. Protein concentrations were measured with BCA protein assay kit (Thermo Fisher Scientific). ALKBH5 (HPA007196) and METTL14 (HPA038002) antibodies were from Sigma. YTHDF1 (17479-1-AP) and PTGER4 (66921-1-Ig) antibodies were from Proteintech. METTL3 (ab195352), FTO (ab92821), and Tenascin C (ab108930) antibodies were from Abcam. WNK1 (MA5-35466) and NLRP12 (PA5-89879) antibodies were from Invitrogen. GAPDH (M171-3) and YTHDF2 (RN123PW) antibodies were from MBL. Lamin A/C (4777 S) antibody was from CST. Goat anti-rabbit IgG-HRP (ZB-2301) and goat anti-mouse IgG-HRP (ZB-2305) antibodies were from ZSGB-BIO.

Liu Y., Song R., Zhao L., Lu Z., Li Y., Zhan X., Lu F., Yang J., Niu Y, & Cao X. (2022). m6A demethylase ALKBH5 is required for antibacterial innate defense by intrinsic motivation of neutrophil migration. Signal Transduction and Targeted Therapy, 7, 194.

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Cell and Tissue Lysis for Proteomic Analysis

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After 48h post infection, the adipocytes were washed four times with phosphate buffered saline (pH 7.4) and lysed in 1 ml lysis buffer containing 50 mmol/l Tris pH 7.5, 1% NP-40, and 150 mmol/l sodium chloride plus protease inhibitor cocktail. Adipose tissues were homogenized in TNET buffer lacking Triton X-100 (150 mm NaCl, 5 mm EDTA, 50 mm Tris-HCl, pH 7.5), supplemented with complete protease inhibitor cocktail and phosphatase inhibitor cocktail (Thermofisher). This was followed by low-speed centrifugation (3000 × g at 4° C) to remove the fat cake from the top of the tube. Triton X-100 was added to the homogenate for a final concentration of 1%, and the extract was cleared at 20,000 × g for 15 min at 4° C and mixed with 2× Laemmli sample buffer. The lung tissues were lysed in RIPA buffer (150 mm NaCl, 0.1% SDS, 1% triton X-100, 2 mM EDTA, 1% sodium deoxycholate, Tris-HCl pH 7.5) supplemented with complete protease inhibitor cocktail and phosphatase inhibitor cocktail (Thermofisher) and centrifuged at 12,000 rpm for 15 min at 4° C.

Ayyappan J.P., Vinnard C., Subbian S, & Nagajyothi J.F. (2017). Effect of Mycobacterium tuberculosis Infection on Adipocyte Physiology. Microbes and infection, 20(2), 81-88.

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Isolation and Fractionation of Rat Brain Synaptosomal Membranes

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After drug administration, rats were anesthetized with equithesin (5 ml/kg, i.p.) and were decapitated. Rat brains were removed and cut into coronal slices (600–800 μm). The entire striatum containing the caudate putamen and nucleus accumbens and hippocampus were removed at 4°C. To isolate hippocampal subfields, Cornu Ammonis area 1 (CA1), the bend region of CA3, and the dentate gyrus (DG) region centered by the dentate hilus were separated from the slices by microdissection (Zhao et al., 2001 (link)). The dissected hippocampal and striatal tissues were homogenized in isotonic sucrose homogenization buffer (0.32 M sucrose, 10 mM HEPES, pH 7.4, and 2 mM EDTA) containing a protease inhibitor cocktail and a phosphatase inhibitor cocktail (Thermo Scientific, Rochester, NY). The homogenate was centrifuged at 800 g (10 min). The supernatant was centrifuged at 10,000 g (30 min). The pellet 2 (P2) containing crude synaptosomal plasma membranes was washed and centrifuged again at 10,000 g (30 min). The supernatant was removed and the pellet (synaptosomal membranes) was resuspended and solubilized in the sucrose homogenization buffer with 0.5% Triton X-100, 1% sodium dodecyl sulfate (SDS), a protease inhibitor cocktail (Thermo Scientific), and a phosphatase inhibitor cocktail (Thermo Scientific). Protein concentrations were determined. Samples were stored at −80°C until use.

Mao L.M., Xue B., Jin D.Z, & Wang J.Q. (2015). Dynamic increases in AMPA receptor phosphorylation in the rat hippocampus in response to amphetamine. Journal of neurochemistry, 133(6), 795-805.

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Immunoprecipitation and Mass Spectrometry

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Mouse brain tissues were lysed with IP standard lysis buffer (Thermo Fisher scientific, USA) with protease and a phosphatase inhibitor cocktail (1:1000, Thermo Fisher scientific, USA) for 30 min at 4 °C and centrifuged for 15 min at 12,000 rpm. The supernatant was subjected to immunoprecipitation with the indicated antibodies on a rotator for 4 h at 4 °C. Then, 40 μl of agarose-conjugated protein G beads (Roche, Basel, Switzerland) was added, and the mixture was incubated at 4 °C overnight. On the next day, the mixture was washed 5 times (5 min each time) with pre-cooled PBS containing protease and a phosphatase inhibitor cocktail (Thermo Fisher Scientific, USA). Finally, the proteins on the beads were denatured with 1% SDS and the supernatant was subjected to immunoblotting or mass spectrometry (MS) determination.

Zhou H., Zhou M., Hu Y., Limpanon Y., Ma Y., Huang P., Dekumyoy P., Maleewong W, & Lv Z. (2021). TNF-α Triggers RIP1/FADD/Caspase-8-Mediated Apoptosis of Astrocytes and RIP3/MLKL-Mediated Necroptosis of Neurons Induced by Angiostrongylus cantonensis Infection. Cellular and Molecular Neurobiology, 42(6), 1841-1857.

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Synaptic Membrane Fractionation from Rat Brain

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HPC were homogenized in 0.32 M ice-cold sucrose containing TRIS 0.01 M, 0.1 mM EGTA, 2 ul/ml protease inhibitors (Sigma-Aldrich), phosphatase inhibitor cocktail (Thermo FisherScientific, Milan, Italy), pH 7.4. 200 μl of homogenate were aliquoted and immediately frozen.
Purified synaptic terminals (synaptosomes) were freshly obtained by centrifugation on discontinuous Percoll gradients as previously reported (Treccani et al., 2014) . For the preparation of synaptic membranes, synaptosomes were resuspended in lysis buffer: 120 mM NaCl, 20 mM HEPES pH 7.4, 0.1 mM EGTA, 0.1 mM DTT, containing 2 ul/ml protease inhibitors (Sigma-Aldrich) and phosphatase inhibitor cocktail (Thermo FisherScientific), pH 7.4. The synaptic membrane fraction was obtained by centrifugation at 29,000 g for 30′ at 4 °C as described previously (Musazzi et al., 2010 ) and stored at -80 °C.

Elhussiny M.E.A., Carini G., Mingardi J., Tornese P., Sala N., Bono F., Fiorentini C., La Via L., Popoli M., Musazzi L, & Barbon A. (2021). Modulation by chronic stress and ketamine of ionotropic AMPA/NMDA and metabotropic glutamate receptors in the rat hippocampus. Progress in neuro-psychopharmacology & biological psychiatry, 104.

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Protein Extraction and Western Blot Analysis

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Total cell extracts were prepared using lysis buffer (20 mM HEPES [pH 7.4], 1% Triton X-100, 1 mM EDTA, 1 mM MgCl₂, 150 mM NaCl, 10% glycerol, protease inhibitor, and phosphatase inhibitor cocktail [Invitrogen]). Protein concentrations were determined using micro-BCA protein reagent (Pierce Biotechnology). Thirty micrograms of total proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes with 0.2-μm pore size (Whatman). The membranes were incubated with antibodies against phospho-AKT (Ser473) (#4060, 1:1000; Cell Signaling Technology (CST); RRID: AB_2315049), AKT (#9272, 1:1000; CST; RRID: AB_329827), phospho-mTOR (Ser2448) (#2971, 1:1000; CST; RRID: AB_330970), mTOR (#2972, 1:1000; CST; RRID: AB_330978), phospho-S6 ribosomal protein (Ser2235/236) (#2211, 1:1000; CST; RRID: AB_331679), S6 ribosomal protein (#2217, 1:1000; CST; RRID: AB_331355), phospho-4E-BP1 (Thr70) (#13396, 1:1000; CST; RRID: AB_2798206), 4E-BP1 (#9644, 1:1000; CST; RRID: AB_2097841), phospho-EGFR (Tyr1068) (#3777, 1:1000; CST; RRID: AB_2096270), EGFR (#2646, 1:1000; CST; RRID: AB_2230881), RNF11 (ab154831, 1:1000; Abcam), or β-actin (AC-15, 1:5000; Sigma; RRID: AB_476692). The ECL method (Invitrogen) was used for protein detection.

Sa J.K., Hong J.Y., Lee I.K., Kim J.S., Sim M.H., Kim H.J., An J.Y., Sohn T.S., Lee J.H., Bae J.M., Kim S., Kim K.M., Kim S.T., Park S.H., Park J.O., Lim H.Y., Kang W.K., Her N.G., Lee Y., Cho H.J., Shin Y.J., Kim M., Koo H., Kim M., Seo Y.J., Kim J.Y., Choi M.G., Nam D.H, & Lee J. (2020). Comprehensive pharmacogenomic characterization of gastric cancer. Genome Medicine, 12, 17.

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Regulation of EMT Markers by NNT-AS1 in CRC

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CRC cells (SW480 and SW620) were transfected with NNT-AS1 siRNAs or Control siRNA in 6-well plates. Seventy-two hours after transfection, the cells were washed twice with cold PBS, and 30 μL of RIPA (Solarbio, Shanghai, China) containing a protease inhibitor cocktail (Invitrogen, Carlsbad, CA, USA), a phosphatase inhibitor cocktail (Invitrogen, Carlsbad, CA, USA) and 2 mM ethylenediaminetetraacetic acid (EDTA) at pH 8.0 was added to each well. Following this, the cells were collected into a cold tube. Cell lysates were centrifuged at 12,000 g for 20 min at 4°C. Subsequently, the proteins in the lysates were separated on a 10% polyacrylamide gel and transferred onto polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 10% non-fat milk for 4 h at room temperature and then incubated with primary antibodies for 1 h at 4°C. Antibodies against the following proteins were used: E-cadherin (24E10), vimentin (D21H3), p44/42 MAPK (Erk1/2), phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E), GAPDH (D16H11) (from Cell Signaling Technology, Danvers, MA) and Ras [EP1125Y] (from Abcam, Cambridge, UK), followed by second antibody (zhongshanjinqiao, China). Protein bands were visualized using Super Enhanced Chemiluminescence Detection regents (Applygen Technologies, Beijing, China).

Wang Q., Yang L., Hu X., Jiang Y., Hu Y., Liu Z., Liu J., Wen T., Ma Y., An G, & Feng G. (2016). Upregulated NNT-AS1, a long noncoding RNA, contributes to proliferation and migration of colorectal cancer cells in vitro and in vivo. Oncotarget, 8(2), 3441-3453.

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Neuronal Proteome Labeling and Analysis

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Neurons were grown and treated with various agents (proteasome inhibitors and other inhibitors) in Neurobasal A (Invitrogen). After treatment, the medium was changed to Neurobasal A lacking Methionine and supplemented with 4 mM AHA + / - treatments for 10 min. Neurons were washed 3x with PBS and lysed in PBS with 1% (w/v) Triton X100, 0.4% (w/v) SDS, protease inhibitors w/o EDTA (Calbiochem, 1:750) and benzonase (Sigma, 1:1000), heated at 95°C and centrifuged. BONCAT was performed as described in Dieterich et al. (2006) (link). In brief, 30 µg of total protein was subjected to a click reaction with 300 μM Triazol (Sigma, ref 678937), 50 μM biotin-alkyne tag (Thermo, ref B10185) and 83 μg/mL CuBr at 4°C o.n. in the dark. Biotinylated proteins were detected by Immunoblot. For puromycylation, puromycin was added to the culture media + / - treatments to a final concentration of 1.5 ng/µl for 10 min, then the neurons were washed 2x with PBS and lysed. For phosphorylation studies a phosphatase inhibitor cocktail (Invitrogen) was added to the lysis buffer. Tissue from HRI KO or wild-type mice was homogenized in lysis buffer (1% (w/v) Triton X100, 1% (w/v) SDS, protease inhibitors (1:750) and benzonase (1:500), heated to 95°C and centrifuged. SDS-PAGE was performed with 3 µg of lysate and proteins were subsequently blotted onto PVDF membranes.

Alvarez-Castelao B., tom Dieck S., Fusco C.M., Donlin-Asp P., Perez J.D, & Schuman E.M. (2020). The switch-like expression of heme-regulated kinase 1 mediates neuronal proteostasis following proteasome inhibition. eLife, 9, e52714.

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Akt Phosphorylation Induction in EPCR-Deficient Cells

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Induction of Akt phosphorylation on EA.hy926 EPCR^KD was performed as described with minor modifications (19 (link)). EA.hy926 EPCR^KD cells were grown to confluence in 12-well dishes followed by incubation with 20 μg/mL EPCR-GPI in serum-free medium (SFM) for 2 hours. After incubation, cells were washed once with SFM and cells were starved over-night in 2% serum containing medium. After treatment with APC (50 nM) cell lysates were made with 100 μL of 1.5% NP-40 lysis buffer containing 1× protease inhibitor cocktail (Pierce) and 1× phosphatase inhibitor cocktail (Invitrogen). Lysates were centrifuged (30 min, 14000 rpm), mixed with SDS sample buffer (LI-COR) and separated on 4–12% SDS Nu-PAGE (Invitrogen). Proteins were transferred to Nitrocellulose membrane (Thermo Scientific), blocked with Odyssey Blocking Buffer (LI-COR) and incubated with primary antibodies (1/2000) against Akt and phospho-Ser473 Akt (Cell Signaling). Blots were developed with donkey anti-mouse IRDye 680 (total Akt) and donkey anti-rabbit IRDye 800CW (phospho-Akt) secondary antibodies (LI-COR) and scanned on the Odyssey Imager (LI-COR). Quantification of integrated fluorescence intensity (K counts) was done using Odyssey Application Software v3.0 (LI-COR).

Bouwens E.A., Stavenuiter F, & Mosnier L.O. (2015). Cell painting with an engineered EPCR to augment the protein C system. Thrombosis and haemostasis, 114(6), 1144-1155.

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