CML and AML cell lines were cultured in the presence or absence of OP449. Following the indicated drug exposure time, cells were washed in PBS and lysed in 50 μL lysis buffer (Cell Signaling, Boston, MA) supplemented with complete protease inhibitor and phosphatase inhibitor cocktail-2 (Sigma-Aldrich, St. Louis, MO). Equal amounts of protein were fractionated on 4-15% Tris-glycine polyacrylamide gels (Bio-Rad, Hercules, CA), transferred to PVDF membranes, and probed with the indicated antibodies: BCR, pABL, STAT5, pSTAT5, AKT, pAKT (S473), pAKT (T308), ERK1/2, pERK1/2, P38/MAPK, pP38/MAPK, S6, pS6 ribosomal (S235/236) (Cell Signaling), SET, PP2Ac (Millipore), and α-tubulin (Sigma-Aldrich).
Pp2ac
Manufactured by Merck Group
Sourced in United States
PP2Ac is a laboratory product manufactured by Merck Group. It is a purified protein phosphatase 2A catalytic subunit. PP2Ac is a key regulatory enzyme involved in various cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
α tubulin, by Merck Group (2 mentions)
Flag m2, by Merck Group (2 mentions)
β catenin, by BD (2 mentions)
P p38 mapk, by Cell Signaling Technology (1 mentions)
P erk1 2, by Cell Signaling Technology (1 mentions)
Complete protease inhibitor and phosphatase inhibitor cocktail 2, by Merck Group (1 mentions)
Tris glycine polyacrylamide gel, by Bio-Rad (1 mentions)
P38 mapk, by Cell Signaling Technology (1 mentions)
Erk1 2, by Cell Signaling Technology (1 mentions)
A3682, by Merck Group (1 mentions)
Calsequestrin, by Dianova (1 mentions)
Pp 1α, by Santa Cruz Biotechnology (1 mentions)
Empagliflozin, by Selleck Chemicals (1 mentions)
Dapagliflozin, by Selleck Chemicals (1 mentions)
Phloretin, by Merck Group (1 mentions)
Lamin b, by Cell Signaling Technology (1 mentions)
Fty720, by Merck Group (1 mentions)
Canagliflozin, by Selleck Chemicals (1 mentions)
Glut3, by Merck Group (1 mentions)
Mouse igg2b apc, by Miltenyi Biotec (1 mentions)
12 protocols using pp2ac
1
Signaling Pathway Analysis in Leukemia Cells
2
Cardiac Protein Phosphorylation Analysis
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After incubation with H2O2, NRCMs were lysed with ice-cold GST-fish buffer (10% glycerol, 50 mM Tris pH 7.4, 150 mM NaCl, 1% NP-4, 4 mM MgCl2, 1% IGEPAL CA-630, complete protease inhibitor), cell debris was removed at 13,000 g (10 min, 4 °C). For non-reducing 12–15% acrylamide SDS-PAGE, the cells were lysed in the presence of 10 mM maleimide but without DTT and heat. Gels were blotted onto a nitrocellulose membrane, blocked with 1x Roti-Block (Roth) for 1 h, washed with TBST (Tris-buffered saline, 0.1% Tween 20) and incubated overnight at 4 °C with a primary antibody: calsequestrin (1:1000, Source: rabbit, Dianova, ABR-01164), PP-1α (1:200, Source: goat, Santa-Cruz, sc-6104), PP-2Ac (1:1000, Source: rabbit, Millipore, 07–324), PKA-RI (1:200, Source: mouse, BD Transduction laboratories, 610166), PLB-pSer16 (1:5000, Source: rabbit, Badrilla Ltd., A010–12), cMyBPC-pSer282 (1:5000, Source: rabbit, Enzo Life Science, ALX-215–057-R050), and I-1-pThr35 (1:1000, Source: rabbit, Cell signalling, #2302). The membrane was washed three times with TBST for 10 min each and incubated for 1 h with the secondary antibody: HRP-coupled antibodies (mouse: Sigma-Aldrich, A3682; rabbit: Sigma-Aldrich, A0545; goat: Santa-Cruz, sc-2020).
3
Phospho-β-catenin Signaling Pathway Modulation
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Canagliflozin, empagliflozin, and dapagliflozin were purchased from Selleck Chemicals (Houston, TX). MG-132, lithium chloride (LiCl), FTY720, WZB117, and phloretin were obtained from Sigma-Aldrich (St. Louis, MO) and Okadaic acid (OA) was from Cayman Chemical (Ann Arbor, MI). For immunoblotting, antibodies against phospho-β-catenin (Ser33/Ser37/Thr41), phospho-β-catenin (Ser45), β-catenin, Cyclin D1, Tubulin, Lamin B, and DDK-tag were purchased from Cell Signaling (Danvers, MA), and Glut1 and GAPDH antibodies were from Abcam (Cambridge, MA). PP2Ac, Glut3, and Actin antibodies were obtained from Millipore (Billerica, MA), Santa Cruz Biotechnology (San Diego, CA), and ProteinTech (Rosemont, IL), respectively. For experiments related to flow cytometry, Monoclonal CD133/2 (293C3)-APC and Mouse-IgG2b-APC antibodies were purchased from Miltenyi Biotech (Bergish Gladbach, Germany), and FITC anti-human CD326 (EpCAM) and Mouse-IgG2b-FITC antibodies were from BioLegend (San Diego, CA).
4
Western Blot Analysis of β-Catenin Signaling
5
Coimmunoprecipitation and Western Blot Analyses
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Tissue proteins were extracted in IP Lysis Buffer (Thermo Fisher Scientific) mixed with cOmplete Protease Inhibitor Cocktail (Roche), and lysates were incubated overnight at 4°C with 5 μg anti-ERα (MC20; Santa Cruz Biotechnology) or anti-striatin (BD Bioscience) antibody. Next, the lysates were incubated with protein G beads (Amersham Biosciences) for 2 h at 4°C, and the pellets obtained after centrifugation were washed five times and analyzed by immunoblotting. Proteins were resolved by dodecyl sulfate-PAGE, transferred to polyvinylidene fluoride membranes, and probed with the appropriate primary antibodies, including AMPK, GAPDH (Santa Cruz Biotechnology), PP2Ac (Millipore), phospho (p)-AMPK, p-Akt, total Akt (Cell Signaling Technology), and α-tubulin (EMD). Membranes were then incubated with the appropriate secondary antibodies and developed using ECL Prime (Amersham Biosciences). The Proteome Profiler Antibody Array (R&D Systems) was used for phospho-kinase analysis, according to the manufacturer’s instructions.
6
Immunoprecipitation of ARPP19 Complexes
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Synthetic DNA encoding the various ARPP19 sequences was purchased from GeneArt, Life Technologies and cloned into the pcDNA5/FRT/TO (Invitrogen) expression vector containing YFP resulting in YFP–ARPP19 fusion proteins. These constructs were transiently transfected into HeLa cells 24 h prior to collecting cells. Cells were lysed in lysis buffer (50 mM Tris-HCl pH 7.5, 50 mM NaCl, 1 mM EDTA, 1 mM DTT and 0.1% NP40). Complexes were immunoprecipitated at 4 °C in lysis buffer with GFP-Trap (ChromoTek) beads as described by the manufacturer. Precipitated protein complexes were washed 3 times in lysis buffer, eluted in 2× SDS sample buffer and subjected to western blotting using the following antibodies: YFP (1:5,000; generated in house), B55α (1:2,000; 5689S, Cell Signaling Technology), PP2Ac (1:2,000; 05-421, Millipore). Uncropped blots are shown in Supplementary Fig. 1 .
7
Protein Interaction Profiling via IP-Western
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Cells were lysed in a buffer containing 50 mmol/L Tris-HCl, pH 7.5, 150 mmol/L NaCl, 1 mmol/L EDTA, a protease inhibitor cocktail, and 0.3% CHAPS. Cell lysates were incubated overnight at 4°C with the indicated antibodies with constant shaking prior to incubation with protein A/G magnetic beads. Then, the samples were spun down for 1 min and the pelleted beads were washed sequentially with PBS with 1% Triton-X-100, PBS with 0.5% Triton-X-100, and PBS with 0.1% Triton-X-100. The pellets were subsequently re-suspended in SDS sample buffer and subjected to Western blotting. The antibodies used in these experiments included PP2A/A (Millipore, Darmstadt, Germany), PP2A/B (Millipore), PP2A/B’ (Millipore), PP2A/C (Millipore), PyMT (Abcam), endoglin (Abcam), ERK 1/2(Abcam), phospho-ERK 1/2 (Abcam), AKT (Abcam) and phospho-AKT (Abcam).
8
Antibody Panel for Signaling Pathway Analysis
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The following antibodies were used: PR65 (sc-15355, Santa Cruz, CA, USA), PP2Ac (05-421, Millipore), FLAG M2 (F1804, Sigma Aldrich), HA (sc-7392, Santa Cruz), phospho-AKT (Thr308) (9275, Cell Signaling), phospho-AKT (Ser473) (9271, Cell Signaling), phospho-SRC (Tyr416) (6943, Cell Signaling), phospho-FAK (Tyr397) (3283, Cell Signaling), phospho-PP2Ac (Tyr307) (ab32104, Abcam), SRC (2108, Cell Signaling), p21 (2946, Cell Signaling), β-actin (sc-4778, Santa Cruz), phospho-SAPK/JNK (Thr183, Tyr185) (9251, Cell Signaling), phospho-c-Jun (Ser73) (9164, Cell Signaling), phospho-c-Jun (Ser63) (9261, Cell Signaling), and c-Jun (sc-1694, Santa Cruz).
9
Immunoprecipitation and Phosphatase Assay of PP2A
10
Co-immunoprecipitation of Synapsin and PP2Ac
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The procedure of co-immunoprecipitation was previously described (Sun et al., 2009 (link)). Briefly, equal amounts of protein extracts (250 µg) were pooled from 2 to 3 rats per group and incubated in RIPA lysis buffer containing antibody against total synapin (9 µg; Cell Signaling) or PP2Ac (8 µg; Millipore) overnight, followed by the addition of protein A/G agarose beads (30 µL; Santa Cruz Technologies) for 4 hours at 4°C. Beads were then washed 4 times in the RIPA lysis buffer. After final centrifugation, Lammeli buffer containing 1% β-mercaptoethanol was added, boiled for 5 minutes, loaded into gels, and analyzed by immunoblotting procedures as described above. Two percent of protein extract was used as input to normalize the protein band intensity.
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