Journal: International Journal of Molecular Medicine

Article Title: Protein phosphatase 2A interacts with Chk2 and regulates phosphorylation at Thr-68 after cisplatin treatment of human ovarian cancer cells

doi: 10.3892/ijmm.17.5.703

Figure Lengend Snippet: Figure 1. Existence of Chk2-PP2A complex. (A) Immunostaining to locate sites of association between the two enzymes. A2780 cells were treated with 3 μM cisplatin for 1 h and double stained with anti-p-Chk2 antibodies (left, green) or anti-PP2A antibodies (middle, red) 16 h later. Colocalization of the two images is seen where the signal appears as yellow spots in the right panel. (B) Coimmunoprecipitation of PP2A with Chk2 and with HA-Chk2. Immunoprecipitations from cisplatin-treated A2780 cell extracts were performed with antibodies of anti-Chk2 (Chk2) or anti-normal mouse serum (NMS) (a). HA-Chk2 immunoprecipitates were obtained with anti-HA antibody from cells transfected with wild-type plasmid of HA-hChk2 pEFBOS and treated with cisplatin (b). Immune complexes were subjected to immunoblot analyses with anti-p-Chk2(Thr68), anti-Chk2, anti-PP2A-A, anti-PP2A-B and anti-PP2A-C antibodies.

Article Snippet: PP2A and p-Chk2 were visualized using secondary antibodies, fluorescein goat antirabbit conjugate for p-Chk2 and fluorescein goat anti-mouse IgG conjugate for PP2A (Molecular Probes, Invitrogen Life Technologies) at 1:200 for 1 h at 4 ̊C.

Techniques: Immunostaining, Staining, Transfection, Plasmid Preparation, Western Blot

Journal: International Journal of Molecular Medicine

Article Title: Protein phosphatase 2A interacts with Chk2 and regulates phosphorylation at Thr-68 after cisplatin treatment of human ovarian cancer cells

doi: 10.3892/ijmm.17.5.703

Figure Lengend Snippet: Figure 2. (A) Cisplatin-induced Chk2 phosphorylation at Thr-68. A2780 ovarian cancer cells were treated with cisplatin (CDDP) at the IC50 dose (3 μM) for 1 h. The drug was removed by washing cells with PBS. Cells were then incubated in fresh drug-free media for the indicated hours (0, 3, 6, 12, 24 and 48) until harvest. Proteins were extracted and separated on SDS-PAGE gels. Blots were probed with antibodies of anti-p-Chk2 or anti- Chk2, or anti-ß-actin as a control. (B) Quantification of p-Chk2 expression was determined using IPLab-Gel software. (C) siRNA-mediated PP2A silencing affects the p-Chk2 level in A2780 cells. Cells were transfected with siRNA to the catalytic subunit of PP2A for 24 h and treated with 3 μM cisplatin for 1 h. The extent of phosphorylated Chk2 and p53, and the protein level of Chk2, PP2A and ß-actin were estimated using antibodies against respective p-Chk2(Thr68), p-p53(Ser15), Chk2, PP2A and ß-actin.

Article Snippet: PP2A and p-Chk2 were visualized using secondary antibodies, fluorescein goat antirabbit conjugate for p-Chk2 and fluorescein goat anti-mouse IgG conjugate for PP2A (Molecular Probes, Invitrogen Life Technologies) at 1:200 for 1 h at 4 ̊C.

Techniques: Phospho-proteomics, Incubation, SDS Page, Control, Expressing, Software, Transfection

Journal: International Journal of Molecular Medicine

Article Title: Protein phosphatase 2A interacts with Chk2 and regulates phosphorylation at Thr-68 after cisplatin treatment of human ovarian cancer cells

doi: 10.3892/ijmm.17.5.703

Figure Lengend Snippet: Figure 3. (A) Inhibition of PP2A activity by okadaic acid enhances Chk2 phosphorylation. A2780 cells were pretreated with concentrations of OA (0, 5, 10 and 20 nM) for 30 min and then treated with 3 μM cisplatin for 1 h under continuous exposure to OA. Cells were washed to remove cisplatin and re-incubated for 24 h in a replacement of fresh media containing OA. Proteins were prepared, separated and probed with antibodies of anti-p-Chk2, anti-Chk2, or anti-ß-actin. ß-actin served as a control. (B) Inhibitory effects of okadaic acid on protein phosphatase activity by protein phosphatase activity assay. Cells were pretreated with okadaic acid and then treated with cisplatin as described above. The protein phosphatase activity assay was performed following the manufacturer's instructions. Results are expressed as the means ± SD (n=3).

Article Snippet: PP2A and p-Chk2 were visualized using secondary antibodies, fluorescein goat antirabbit conjugate for p-Chk2 and fluorescein goat anti-mouse IgG conjugate for PP2A (Molecular Probes, Invitrogen Life Technologies) at 1:200 for 1 h at 4 ̊C.

Techniques: Inhibition, Activity Assay, Phospho-proteomics, Incubation, Control, Phosphatase Assay

Journal: International Journal of Molecular Medicine

Article Title: Protein phosphatase 2A interacts with Chk2 and regulates phosphorylation at Thr-68 after cisplatin treatment of human ovarian cancer cells

doi: 10.3892/ijmm.17.5.703

Figure Lengend Snippet: Figure 4. Direct dephosphorylation of p-Chk2 by PP2A in vitro. Chk2 was phosphorylated by cisplatin treatment to the A2780 cells. Forty μg of whole cell lysates (A), Chk2-immunoprecipitated protein (B), or HA-Chk2 immunoprecipitated protein (C) was incubated with 0.05 or 0.5 units of PP2A enzyme in the presence or absence of the phosphatase inhibitor OA (B and C), which was used to verify the specificity of the PP2A effect.

Article Snippet: PP2A and p-Chk2 were visualized using secondary antibodies, fluorescein goat antirabbit conjugate for p-Chk2 and fluorescein goat anti-mouse IgG conjugate for PP2A (Molecular Probes, Invitrogen Life Technologies) at 1:200 for 1 h at 4 ̊C.

Techniques: De-Phosphorylation Assay, In Vitro, Immunoprecipitation, Incubation

Journal: The Journal of biological chemistry

Article Title: Positive regulation of Raf1-MEK1/2-ERK1/2 signaling by protein serine/threonine phosphatase 2A holoenzymes.

doi: 10.1074/jbc.M502464200

Figure Lengend Snippet: FIGURE 2. RNAi-mediated knock-down of the PP2A regulatory subunit B. A, total RNA was isolated from multiple tetracycline-treated HEK T-Rex monoclonal cell lines stably expressing EV, B siRNA (B-KD), or epitope-tagged B subunit (B-FLAG or B-FLAG). Reverse transcription-PCR of the RNA was performed using primers specific for B, B, or GAPDH. Shown are ethidium bromide-stained 1.2% agarose gels of the result- ing PCR products. B, protein extracts (15 g) from the cell lines were analyzed by immunoblotting with B/ and GAPDH antibodies.

Article Snippet: IRDyeTM800- and AlexaFluor 680-conjugated fluorescent secondary antibodies were obtained from Rockland Immunochemicals (Gilbertsville, PA) andMolecularProbes (Eugene,OR), respectively.Commercial sources of other antibodies utilized in these studies were as follows: anti-phosphoRaf1(Ser-259), anti-phospho-MEK1/2(Ser-217 Ser-221), and anti-phospho-ERK1/2 (Thr-202 Tyr-204/Thr-185 Tyr-187) antibodies were from Cell Signaling Technology Inc. (Beverly, MA), anti-Raf1 (sc-133 and sc-7267) and anti-ERK1 (sc-93-G) antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA), anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody was from Abcam, Inc. (Cambridge, MA), and anti-PP2A catalytic subunit antibody was from BD Biosciences Pharmingen.

Techniques: Knockdown, Isolation, Stable Transfection, Expressing, Reverse Transcription, Staining, Western Blot

Journal: The Journal of biological chemistry

Article Title: Positive regulation of Raf1-MEK1/2-ERK1/2 signaling by protein serine/threonine phosphatase 2A holoenzymes.

doi: 10.1074/jbc.M502464200

Figure Lengend Snippet: FIGURE 4. Activation of MEK1/2 and ERK1/2 in cells overexpressing PP2A B subunits. A, HEK T-Rex cells stably expressing EV, B-FLAG, or B-FLAG were treated with tetracycline for 24 h, serum-starved overnight, and then stimulated with EGF (50 ng/ml) for the indicated time points. Cell lysates (15 g of protein) were resolved by SDS-PAGE and immunoblotted with antibodies recognizing B/, p-ERK1/2, total ERK1/2, p-MEK1/2, and total MEK1/2. The data are repre- sentative of experiments that were performed at least three times. B, graphical representation of the data shown in A. The p-ERK1/2 and p-MEK1/2 signals were normalized to total ERK1/2 and MEK1/2 levels, respectively; the -fold activations are relative to normalized values of unstimulated cells expressing EV, which were set as 1.

Article Snippet: IRDyeTM800- and AlexaFluor 680-conjugated fluorescent secondary antibodies were obtained from Rockland Immunochemicals (Gilbertsville, PA) andMolecularProbes (Eugene,OR), respectively.Commercial sources of other antibodies utilized in these studies were as follows: anti-phosphoRaf1(Ser-259), anti-phospho-MEK1/2(Ser-217 Ser-221), and anti-phospho-ERK1/2 (Thr-202 Tyr-204/Thr-185 Tyr-187) antibodies were from Cell Signaling Technology Inc. (Beverly, MA), anti-Raf1 (sc-133 and sc-7267) and anti-ERK1 (sc-93-G) antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA), anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody was from Abcam, Inc. (Cambridge, MA), and anti-PP2A catalytic subunit antibody was from BD Biosciences Pharmingen.

Techniques: Activation Assay, Stable Transfection, Expressing, SDS Page

Journal: The Journal of biological chemistry

Article Title: Positive regulation of Raf1-MEK1/2-ERK1/2 signaling by protein serine/threonine phosphatase 2A holoenzymes.

doi: 10.1074/jbc.M502464200

Figure Lengend Snippet: FIGURE 8. PP2A subunits co-immunoprecipitate with Raf1. Cells expressing EV, B-FLAG, B-FLAG, or B siRNA (B-KD) were unstimulated (A) or treated with or without EGF (B) prior to lysis in buffer B. A, FLAG immune complexes (FLAG IPs) were isolated from the clarified cell lysates and subjected to Western analysis using Raf1, FLAG, and PP2Ac antibodies. B, Raf1 immune complexes (Raf1 IPs) from lysates of cells treated with () or without () EGF were immunoblotted for Raf1, FLAG-B, and PP2Ac. An aliquot of each cell lysate was also probed for Raf1 and the indicated PP2A subunits using the corresponding antibodies (A and B, bottom panels).

Article Snippet: IRDyeTM800- and AlexaFluor 680-conjugated fluorescent secondary antibodies were obtained from Rockland Immunochemicals (Gilbertsville, PA) andMolecularProbes (Eugene,OR), respectively.Commercial sources of other antibodies utilized in these studies were as follows: anti-phosphoRaf1(Ser-259), anti-phospho-MEK1/2(Ser-217 Ser-221), and anti-phospho-ERK1/2 (Thr-202 Tyr-204/Thr-185 Tyr-187) antibodies were from Cell Signaling Technology Inc. (Beverly, MA), anti-Raf1 (sc-133 and sc-7267) and anti-ERK1 (sc-93-G) antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA), anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody was from Abcam, Inc. (Cambridge, MA), and anti-PP2A catalytic subunit antibody was from BD Biosciences Pharmingen.

Techniques: Expressing, Lysis, Isolation, Western Blot

Journal: The Journal of biological chemistry

Article Title: Positive regulation of Raf1-MEK1/2-ERK1/2 signaling by protein serine/threonine phosphatase 2A holoenzymes.

doi: 10.1074/jbc.M502464200

Figure Lengend Snippet: FIGURE 9. PP2A-mediated dephosphorylation of Raf1. A, Raf1 immune complexes from cells treated with () or without () EGF were prepared in buffer A and then washed three times in phosphatase assay buffer containing or lacking 500 nM microcys- tin-LR (MC-LR). Dephosphorylation reactions were initiated by the addition of purified PP2AABCorABCholoenzymes(90ng)thathadbeenpretreatedwith()orwithout () 500 nM microcystin-LR. B and C, Raf1 or ERK1/2 immune complexes isolated from extracts of cells grown under normal conditions were incubated with ABC, ABC, or ABC holoenzymes (90 ng). FLAG eluate from EV-expressing cells was used as a con- trol. Following a 30-min incubation at 37 °C, the phosphatase reactions were terminated by the addition of SDS sample buffer and subjected to Western analysis using antibodies recognizing p-Raf1(Ser-259), total Raf1, p-ERK1/2, total ERK1/2, and PP2Ac.

Article Snippet: IRDyeTM800- and AlexaFluor 680-conjugated fluorescent secondary antibodies were obtained from Rockland Immunochemicals (Gilbertsville, PA) andMolecularProbes (Eugene,OR), respectively.Commercial sources of other antibodies utilized in these studies were as follows: anti-phosphoRaf1(Ser-259), anti-phospho-MEK1/2(Ser-217 Ser-221), and anti-phospho-ERK1/2 (Thr-202 Tyr-204/Thr-185 Tyr-187) antibodies were from Cell Signaling Technology Inc. (Beverly, MA), anti-Raf1 (sc-133 and sc-7267) and anti-ERK1 (sc-93-G) antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA), anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody was from Abcam, Inc. (Cambridge, MA), and anti-PP2A catalytic subunit antibody was from BD Biosciences Pharmingen.

Techniques: De-Phosphorylation Assay, Phosphatase Assay, Purification, Isolation, Incubation, Expressing, Western Blot

Journal: Frontiers in Physiology

Article Title: Activation of Yes-Associated Protein/PDZ-Binding Motif Pathway Contributes to Endothelial Dysfunction and Vascular Inflammation in AngiotensinII Hypertension

doi: 10.3389/fphys.2021.732084

Figure Lengend Snippet: AngII induces YAP/TAZ activation and the expression of pro-inflammatory cytokines via protein phosphatase 2A C (PP2Ac) in HUVECs. (A) AngII increased PP2Ac activity; inhibition of PP2Ac with LB-100 suppressed AngII-mediated enhancement of PP2Ac activity in HUVECs. The protein expressions of PP2Ac (B) and p-PP2Ac (C) ; (D–G) Inhibition of PP2Ac with LB-100 suppressed AngII-induced YAP/TAZ nuclear translocation, the protein expressions of YAP (D) and TAZ (F) in the nucleus fraction, and the protein expressions of p-YAP (E) and p-TAZ (G) in the cytosolic fraction of HUVECs; protein expressions of proinflammatory cytokines MCP-1 (H) and TNFα. (I) * p < 0.05, vs. Ctr group, # p < 0.05, vs. AngII group.

Article Snippet: The membranes were incubated with blocking solution (5% of milk in TBST buffer) at room temperature for 2 h and then incubated with primary antibodies against YAP (Sc-101199, Santa Cruz Biotech., Santa Cruz, CA, United States), p-YAP (13008T, Cell Signaling, Chicago, IL, United States), TAZ (4883S, Cell Signaling, Chicago, IL, United States), p-TAZ (59971S, Cell Signaling, Chicago, IL, United States), PP2Ac (2038S, Cell Signaling, Chicago, IL, United States), p-PP2Ac (Sc-271903, Santa Cruz Biotech., Santa Cruz, CA, United States), monocyte chemoattractant protein (MCP)-1 (Sc-52701, Santa Cruz Biotech., Santa Cruz, CA, United States), tumor necrosis factor (TNF)α (Sc-52746, Santa Cruz Biotech., Santa Cruz, CA, United States), HDAC1 (Sc-81598, Santa Cruz Biotech., Santa Cruz, CA, United States), phosphor-endothelial nitric oxide synthase (p-eNOS, AF3247, Affinity, Jiangsu, China), and GAPDH (60004-1-Ig, Proteintech Group, Danvers, MA, United States) at 4°C overnight (1:500 dilution with blocking solution).

Techniques: Activation Assay, Expressing, Activity Assay, Inhibition, Translocation Assay

Journal: Journal of Biological Chemistry

Article Title: B55α PP2A Holoenzymes Modulate the Phosphorylation Status of the Retinoblastoma-related Protein p107 and Its Activation

doi: 10.1074/jbc.m110.162354

Figure Lengend Snippet: FIGURE 1. p107preferentiallyinteractswithhumantrimericPP2AcomplexesassembledwithB55andtoa lesser extent PR48 (truncated form of PR70) in vitro and in cells. A, GST-pocket proteins pull down PP2A com- plexesfromwholecelllysatesofexponentiallygrowingU-2OScells.300gofwholecelllysatewereincubatedwith comparable amounts of affinity-purified fusion GST-pocket proteins or an excess of GST protein loaded with gluta- thione beads. Supernatants (SN) and bound proteins (Pull-down) were resolved via SDS-PAGE and analyzed by Western blot with antibodies specific for the proteins indicated. GST protein levels were detected via Coomassie Blue staining. B, PP2A/A and PP2A/C were coexpressed with a member of each B subunit family (B55, B562, and PR48)ininsectcellsandtrimericcomplexespurifiedasdescribedpreviously(16).Comparableamountsofthethree trimeric complexes normalized by the amount of PP2A/C in the complex were incubated with glutathione beads loaded with GST-p107 or GST and thoroughly washed. Complexes were resolved by SDS-PAGE and detected by Westernblotanalysis.C,B55andp107interactinU-2OScells.HA-p107orFLAG-p107(F-p107)werecotransfected with HA-B55 in U-2 OS cells. Complexes were immunoprecipitated with -p107, -FLAG, or control antibodies. Complexes were resolved by SDS-PAGE, and proteins were detected with specific antibodies. The star marks two HA-p107 bands detected with -p107 antibodies that were resistant to blotting membrane stripping previous to the -FLAG blot. Relevant proteins are indicated.IPs, immunoprecipitations. D, low levels of endogenous B55 and p107aredetectedinU-2OScells.Complexeswereimmunoprecipitatedwith-p107,-B55,orcontrolantibodies after overnight preclearing of cell lysates (1 mg) with control antibodies. Complexes were resolved via SDS-PAGE, and proteins were detected with -p107, -B55, -PP2A/A, and -PP2A/C antibodies. m-Abs and p-Abs indicate monoclonal and polyclonal antibodies, respectively.

Article Snippet: Antibodies—Anti-p107 (sc-318), anti-pRB (sc-50), anti-HA (sc-805), anti-cyclin A (sc-596), anti-E2F4 (sc-512), anti-p27 (sc-528), anti-B55 (sc-33191), anti-CDK2 (sc-163) rabbit polyclonal antibodies; anti-PP2A/A (sc-6113) and anti-PR48 (sc11801) goat polyclonal antibodies; and anti-HA (sc-7392) and anti-B55 (sc-81606) mouse monoclonal antibody were from Santa Cruz Biotechnology.

Techniques: In Vitro, Affinity Purification, SDS Page, Western Blot, Staining, Incubation, Immunoprecipitation, Control, Membrane, Stripping Membranes

Journal: Journal of Biological Chemistry

Article Title: B55α PP2A Holoenzymes Modulate the Phosphorylation Status of the Retinoblastoma-related Protein p107 and Its Activation

doi: 10.1074/jbc.m110.162354

Figure Lengend Snippet: FIGURE 2. Members of distinct B subunit family target particular pocket proteins. A, B55 interacts with p107 andp130butnotpRBinU-2OScellsectopicallyexpressingtaggedproteins.Thisanalysiswascarriedoutasinpanel B. A cross-reacting band is indicated by an asterisk. IPs, immunoprecipitations. B, U-2 OS cells were transfected with HA-tagged subunits at comparable levels. Pulldown assays were performed with equivalent amounts of the indi- cated GST proteins. Bound B subunits and their input levels were detected via Western blot analysis. Cross-reacting bands are indicated by stars. The asterisk indicates a molecular mass marker co-run in the first well.

Article Snippet: Antibodies—Anti-p107 (sc-318), anti-pRB (sc-50), anti-HA (sc-805), anti-cyclin A (sc-596), anti-E2F4 (sc-512), anti-p27 (sc-528), anti-B55 (sc-33191), anti-CDK2 (sc-163) rabbit polyclonal antibodies; anti-PP2A/A (sc-6113) and anti-PR48 (sc11801) goat polyclonal antibodies; and anti-HA (sc-7392) and anti-B55 (sc-81606) mouse monoclonal antibody were from Santa Cruz Biotechnology.

Techniques: Transfection, Western Blot, Marker

Journal: Journal of Biological Chemistry

Article Title: B55α PP2A Holoenzymes Modulate the Phosphorylation Status of the Retinoblastoma-related Protein p107 and Its Activation

doi: 10.1074/jbc.m110.162354

Figure Lengend Snippet: FIGURE 3. The spacer region of p107 mediates the interaction with the PP2A-B55 complex, and the C terminus enhances the interaction. A, conserved p107 domains and summarized relative binding activity with PP2A (domain designations are defined under “Experimental Procedures”). B, the spacer domain of p107 is sufficient to interact with PP2A/A and PP2A/C, and the C terminus domain of p107 enhances this interaction but fails to interactwithPP2A.Glutathionebeadsloadedwithequivalentmolaramounts of wild type and mutant GST-p107 fusion proteins or GST were incubated with lysates of exponentially growing U-2 OS cells. Complexes were analyzed by Western blot. C, purified PP2A-B55 but not PP2A-B562 holoenzymes interact with the spacer of p107. Pulldown assays were performed with the indicated GST proteins and purified holoenzymes as in Fig. 2A.

Article Snippet: Antibodies—Anti-p107 (sc-318), anti-pRB (sc-50), anti-HA (sc-805), anti-cyclin A (sc-596), anti-E2F4 (sc-512), anti-p27 (sc-528), anti-B55 (sc-33191), anti-CDK2 (sc-163) rabbit polyclonal antibodies; anti-PP2A/A (sc-6113) and anti-PR48 (sc11801) goat polyclonal antibodies; and anti-HA (sc-7392) and anti-B55 (sc-81606) mouse monoclonal antibody were from Santa Cruz Biotechnology.

Techniques: Binding Assay, Activity Assay, Mutagenesis, Incubation, Western Blot, Purification

Journal: Journal of Biological Chemistry

Article Title: B55α PP2A Holoenzymes Modulate the Phosphorylation Status of the Retinoblastoma-related Protein p107 and Its Activation

doi: 10.1074/jbc.m110.162354

Figure Lengend Snippet: FIGURE 4. B55 domains and residues required for binding to p107. A, structure of B55 and residues in its topacidicfacethatareimportantfordephosphorylationoftheB55substrateTau.B,B55Asp-197isessential for the binding of p107. U-2 OS cells were co-transfected with FLAG-p107 and the indicated Myc-B55 con- structs. Cell lysates were immunoprecipitated with -FLAG and -Myc antibodies and resolved via SDS-PAGE. Proteins were detected using -FLAG, -Myc, -PP2A/A, and -PP2A/C antibodies. IP, immunoprecipitates; WB, Western blots.

Article Snippet: Antibodies—Anti-p107 (sc-318), anti-pRB (sc-50), anti-HA (sc-805), anti-cyclin A (sc-596), anti-E2F4 (sc-512), anti-p27 (sc-528), anti-B55 (sc-33191), anti-CDK2 (sc-163) rabbit polyclonal antibodies; anti-PP2A/A (sc-6113) and anti-PR48 (sc11801) goat polyclonal antibodies; and anti-HA (sc-7392) and anti-B55 (sc-81606) mouse monoclonal antibody were from Santa Cruz Biotechnology.

Techniques: Binding Assay, Transfection, Immunoprecipitation, SDS Page, Western Blot

Journal: Journal of Biological Chemistry

Article Title: B55α PP2A Holoenzymes Modulate the Phosphorylation Status of the Retinoblastoma-related Protein p107 and Its Activation

doi: 10.1074/jbc.m110.162354

Figure Lengend Snippet: FIGURE 5. B55 and PR70 mediate an st-sensitive interaction between the PP2A/C-PP2A/A dimer and p107. A and B, SV40 st antigen (small t ag) expression in U-2 OS cells prevents pulldown of PP2A subunits A and C, but not binding to select B subunits (B55 and PR70), by GST-p107. U-2 OS cells were transfected with vectors encoding HA-tagged B subunits (B55, B56, B561, and PR70) and subsequently transduced with adenoviruses encoding st or -galactosidase. GST pulldown assays were performed, and complexes were analyzed by SDS-PAGE followed by Western blot analysis with antibodies raised against the indicated proteins. The effects of st on PP2A complexes are represented schematically. EGFP, enhanced GFP. C, lysates of cells described as in B were immunoprecipitated with -HA antibodies, and the association of B subunits with PP2A/C in the presence or absence of st was determined via Western blot analysis. IP, immunoprecipitates.

Article Snippet: Antibodies—Anti-p107 (sc-318), anti-pRB (sc-50), anti-HA (sc-805), anti-cyclin A (sc-596), anti-E2F4 (sc-512), anti-p27 (sc-528), anti-B55 (sc-33191), anti-CDK2 (sc-163) rabbit polyclonal antibodies; anti-PP2A/A (sc-6113) and anti-PR48 (sc11801) goat polyclonal antibodies; and anti-HA (sc-7392) and anti-B55 (sc-81606) mouse monoclonal antibody were from Santa Cruz Biotechnology.

Techniques: Expressing, Binding Assay, Transfection, Transduction, SDS Page, Western Blot, Immunoprecipitation

Journal: Journal of Biological Chemistry

Article Title: B55α PP2A Holoenzymes Modulate the Phosphorylation Status of the Retinoblastoma-related Protein p107 and Its Activation

doi: 10.1074/jbc.m110.162354

Figure Lengend Snippet: FIGURE 6. Recombinant purified B55 trimeric PP2A complexes dephosphorylate phosphorylated p107 in vitro. A, increasing concen- trations of purified PP2A-B55 complexes were incubated with GST-p107 previously phosphorylated (P) in vitro with purified cyclin A-CDK2 com- plexes. Con, control. Auto-rad, autoradiography. B, comparable amounts of PP2A-B55 and PP2A-B562 purified holoenzymes were incubated with GST-p107 or histone H1 previously phosphorylated with cyclin A-CDK2 complexes.

Article Snippet: Antibodies—Anti-p107 (sc-318), anti-pRB (sc-50), anti-HA (sc-805), anti-cyclin A (sc-596), anti-E2F4 (sc-512), anti-p27 (sc-528), anti-B55 (sc-33191), anti-CDK2 (sc-163) rabbit polyclonal antibodies; anti-PP2A/A (sc-6113) and anti-PR48 (sc11801) goat polyclonal antibodies; and anti-HA (sc-7392) and anti-B55 (sc-81606) mouse monoclonal antibody were from Santa Cruz Biotechnology.

Techniques: Recombinant, Purification, In Vitro, Incubation, Control, Autoradiography

Journal: Journal of Biological Chemistry

Article Title: B55α PP2A Holoenzymes Modulate the Phosphorylation Status of the Retinoblastoma-related Protein p107 and Its Activation

doi: 10.1074/jbc.m110.162354

Figure Lengend Snippet: FIGURE 7. Ectopic expression of B55, but not other subunits, results in st-sensitive dephosphorylation of p107andp130.A,U-2OScellsweretransfectedwiththeindicatedBsubunitsatcomparablelevels,andtheireffect on the phosphorylation of endogenous p107 and p130 was determined via Western blot analysis. Hyper- and hypophosphorylated forms are indicated by hyper-P and hypo-P. B, dose-dependent increase in B55 expression leadstodose-dependentp107dephosphorylation.U-2OScellsweretransfectedasinAwiththeindicatedamounts of plasmid encoding B55. Dephosphorylation was assessed as in A. B55 expression was determined with -HA antibodies. C, U-2 OS cells were transfected as in A and subsequently transduced with either st-expressing or enhanced GFP (EGFP)-expressing adenoviruses. Changes in phosphorylation status were assessed by Western blot analysis. Note that st expression leads to potent hyperphosphorylation of p107. Levels of B55 and st were deter- mined with specific antibodies. Expression of B subunits was determined with -HA antibodies. Hypophosphory- lated p107 and partially hypophosphorylated p130 are marked by an asterisk.

Article Snippet: Antibodies—Anti-p107 (sc-318), anti-pRB (sc-50), anti-HA (sc-805), anti-cyclin A (sc-596), anti-E2F4 (sc-512), anti-p27 (sc-528), anti-B55 (sc-33191), anti-CDK2 (sc-163) rabbit polyclonal antibodies; anti-PP2A/A (sc-6113) and anti-PR48 (sc11801) goat polyclonal antibodies; and anti-HA (sc-7392) and anti-B55 (sc-81606) mouse monoclonal antibody were from Santa Cruz Biotechnology.

Techniques: Expressing, De-Phosphorylation Assay, Phospho-proteomics, Western Blot, Plasmid Preparation, Transfection, Transduction

Journal: Journal of Biological Chemistry

Article Title: B55α PP2A Holoenzymes Modulate the Phosphorylation Status of the Retinoblastoma-related Protein p107 and Its Activation

doi: 10.1074/jbc.m110.162354

Figure Lengend Snippet: FIGURE 8. shRNA-mediated knockdown of B55, but not other B sub- units, results in hyperphosphorylation of p107. U-2 OS cells were trans- fected with the indicated shRNA constructs to B55, B56, and PR70. p107 phosphorylation status and B55 expression were assessed via Western blot analysis. Hyper- and hypophosphorylated forms are indicated by hyper-P and hypo-P.

Article Snippet: Antibodies—Anti-p107 (sc-318), anti-pRB (sc-50), anti-HA (sc-805), anti-cyclin A (sc-596), anti-E2F4 (sc-512), anti-p27 (sc-528), anti-B55 (sc-33191), anti-CDK2 (sc-163) rabbit polyclonal antibodies; anti-PP2A/A (sc-6113) and anti-PR48 (sc11801) goat polyclonal antibodies; and anti-HA (sc-7392) and anti-B55 (sc-81606) mouse monoclonal antibody were from Santa Cruz Biotechnology.

Techniques: shRNA, Knockdown, Construct, Phospho-proteomics, Expressing, Western Blot

Journal: Journal of Biological Chemistry

Article Title: B55α PP2A Holoenzymes Modulate the Phosphorylation Status of the Retinoblastoma-related Protein p107 and Its Activation

doi: 10.1074/jbc.m110.162354

Figure Lengend Snippet: FIGURE 9. Ectopic expression of WT, but not a B55-mutant defective in p107 binding, induces p107 activation. A, ectopic expression of WT, but not a p107-defective B55-mutant, induces p107 dephosphory- lation without altering the expression of E2F-dependent gene products. U-2 OS cells were transduced with retroviruses expressing B55, B55D197K, or no transgene and selected. Whole protein lysates were analyzed by Western blot with specific antibodies. Note that in this particular blot, p130 forms have not been resolved sufficiently to appreciate the relatively less marked dephosphorylation effects (Fig. 7, A and C). Hyper- and hypophosphorylated forms are indicated by Hyper-P and Hypo-P. B, 250 g of whole protein lysates were immunoprecipitated with anti-E2F4 antibodies, and complexes were resolved by SDS-PAGE followed by West- ern blot analysis with antibodies to p107 and p130.

Article Snippet: Antibodies—Anti-p107 (sc-318), anti-pRB (sc-50), anti-HA (sc-805), anti-cyclin A (sc-596), anti-E2F4 (sc-512), anti-p27 (sc-528), anti-B55 (sc-33191), anti-CDK2 (sc-163) rabbit polyclonal antibodies; anti-PP2A/A (sc-6113) and anti-PR48 (sc11801) goat polyclonal antibodies; and anti-HA (sc-7392) and anti-B55 (sc-81606) mouse monoclonal antibody were from Santa Cruz Biotechnology.

Techniques: Expressing, Mutagenesis, Binding Assay, Activation Assay, Transduction, Western Blot, De-Phosphorylation Assay, Immunoprecipitation, SDS Page

Journal: bioRxiv

Article Title: Phosphoprotein Phosphatase Activity Positively Regulates Oligomeric Pyrin to Trigger Inflammasome Assembly in Response to Bacterial Effectors and Toxins that Inactivate RhoA in Macrophages

doi: 10.1101/2022.03.23.485108

Figure Lengend Snippet: siRNA knock down of PP2A in BMDMs reduces pyrin S205 dephosphorylation. WT BMDMs were electroporated with siRNAs targeting PKN1 (400pmol), PP2Aca (1000pmol) or PP2Aca+PP2Acb (500pmol each) as indicated. 24hr after electroporation the BMDMs were LPS-primed. 48hr following electroporation, cells were either left uninfected or infected with ΔyopM Yptb at an MOI of 30 for 90 mins. A and C) RT-qPCR analysis of mRNA transcripts of PP2Aca and PP2Acb in uninfected BMDMs. Results were normalized to Hprt mRNA levels. A) Each data group is presented as an average (error bars are standard deviation) of three independent experiments. One-way ANOVA was applied to calculate significance and p-values as compared to PKN1 control siRNA are indicated. P-value<0.05 was considered significant; <0.0001 (****). C) Results from one experiment are shown. B, and D) Immunoblot analysis of PKN1, PP2A, total and PS205 pyrin, and pro-IL-1β in uninfected and ΔyopM Yptb- infected BMDM lysates. Actin was used as a loading control. E) Mature IL-1β in supernatants as quantified by ELISA. Results represent averages (error bars are standard deviation) from two independent experiments.

Article Snippet: Primary antibodies used in this paper were: rabbit-anti-mouse monoclonal total pyrin antibody (1:1000 dilution, ab195975; abcam), rabbit-anti-mouse pyrin polyclonal (1:1000 dilution, ( )), rabbit-anti-mouse phospho-serine 205 monoclonal antibody (1:1000 dilution, ab201784; abcam), rabbit-anti-mouse phospho-serine 241 monoclonal antibody (1:1000 dilution, ab201784; abcam), rabbit-anti-mouse/human IL-1β (1:1000 dilution, number 12242; Cell Signaling), rabbit-anti-mouse/human polyclonal β-actin (1:1000 dilution, number 4967; Cell Signaling), mouse-anti-mouse/human/rat monoclonal PP1a (1:1000 dilution, number MA5-17239, ThermoFisher Scientific), rabbit-anti- Y.pestis polyclonal YopM (1:1000 dilution, provided by Susan Straley), mouse-anti-mouse monoclonal PKN (1:1000 dilution, number 393344, Santa Cruz Biotechnology), rabbit-anti-mouse monoclonal PP2A (1:1000 dilution, #2038, Cell Signaling), and rabbit-anti-mouse GSDMD (1:1000 dilution, ab209845, Abcam).

Techniques: Knockdown, De-Phosphorylation Assay, Electroporation, Infection, Quantitative RT-PCR, Standard Deviation, Control, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: eLife

Article Title: Division of labour between PP2A-B56 isoforms at the centromere and kinetochore

doi: 10.7554/eLife.42619

Figure Lengend Snippet: ( A ) Schematic, representative images and line plot analysis of B56γ and B56γ EPVA localisation in nocodazole-arrested Flp-in HeLa cells. ( B ) Schematic, representative images and line plot analysis of additional B56γ-α chimaeras to define the extra regions in B56α needed to fully switch B56γ from kinetochores to centromeres. Each graph represents the mean intensities (±SD) from at least 2 independent experiments. 5 kinetochore pairs were analysed per cell, for a total of 10 cells per experiment. Intensity is normalized to the maximum signal in each channel in each experiment. Scale bars, 5µm.

Article Snippet: The following antibodies were used for western blotting (at the final concentration indicated): rabbit α-GFP (custom polyclonal, a gift from G. Kops; 1:5000), mouse α-B56γ (clone A-11, sc-374379, Santa Cruz Biotechnology; 1:1000), mouse α-B56α (clone 23, 610615, BD; 1:1000), mouse α-B56δ (clone H-11, sc-271363, Santa Cruz, 1:1000), rabbit α-B56ε (ARP56694-P050, Aviva, 1:1000), mouse α-PPP2CA (clone 1D6, 05–421, Millipore; 1:5000) and rabbit α-PPP2R1A (clone 81G5, #2041, CST; 1:1000), rabbit α-BubR1 (A300-386A, Bethyl; 1:1000), rabbit α-Axin (C76H11, CST; 1:1000), rabbit α-GEF-H1 (155785, Abcam; 1:1000), rabbit α-Kif4a (A301-074A, Bethyl; 1:1000), rabbit α-RepoMan (HPA030049, Sigma; 1:1000) and rabbit α-Actin (A2066, Sigma; 1:5000) and mouse α-alpha-Tubulin (clone B-5-1-2, T5168, Sigma, 1:5000).

Techniques:

Journal: eLife

Article Title: Division of labour between PP2A-B56 isoforms at the centromere and kinetochore

doi: 10.7554/eLife.42619

Figure Lengend Snippet: B56 localisation in B56α-γ chimaeras spanning the entire B56 (Ch1-4: A–C ), a region at the C-terminus (Ch4a-4d: D–F ). ( A, D ) Schematic representation of the B56α-γ chimaeras created. Representative images ( B, E ) and line plot analysis ( C, F ) to show the B56 localisation pattern in each chimaera. ( G ). Alignment of B56 isoforms within region 4d that controls centromere/kinetochore localisation. ( G–H ): Effect of 4 point-mutations within region 4d to convert B56α to the correspond B56γ sequence (B56α TKHG ). Representative images ( H ) and line plot analysis ( I ) of B56α WT or B56α TKHG in cells arrested in prometaphase with nocodazole. Each graph represents the mean intensities (±SD) from 3 independent experiments. 5 kinetochore pairs were analysed per cell, for a total of 10 cells per experiment. Intensity is normalized to the maximum signal in each channel in each experiment. Scale bars, 5µm.

Article Snippet: The following antibodies were used for western blotting (at the final concentration indicated): rabbit α-GFP (custom polyclonal, a gift from G. Kops; 1:5000), mouse α-B56γ (clone A-11, sc-374379, Santa Cruz Biotechnology; 1:1000), mouse α-B56α (clone 23, 610615, BD; 1:1000), mouse α-B56δ (clone H-11, sc-271363, Santa Cruz, 1:1000), rabbit α-B56ε (ARP56694-P050, Aviva, 1:1000), mouse α-PPP2CA (clone 1D6, 05–421, Millipore; 1:5000) and rabbit α-PPP2R1A (clone 81G5, #2041, CST; 1:1000), rabbit α-BubR1 (A300-386A, Bethyl; 1:1000), rabbit α-Axin (C76H11, CST; 1:1000), rabbit α-GEF-H1 (155785, Abcam; 1:1000), rabbit α-Kif4a (A301-074A, Bethyl; 1:1000), rabbit α-RepoMan (HPA030049, Sigma; 1:1000) and rabbit α-Actin (A2066, Sigma; 1:5000) and mouse α-alpha-Tubulin (clone B-5-1-2, T5168, Sigma, 1:5000).

Techniques: Sequencing

Journal: eLife

Article Title: Division of labour between PP2A-B56 isoforms at the centromere and kinetochore

doi: 10.7554/eLife.42619

Figure Lengend Snippet: Alignment of the BubR1 binding pocket ( A ) and the Sgo1-binding region ( B ) in B56 isoforms.

Article Snippet: The following antibodies were used for western blotting (at the final concentration indicated): rabbit α-GFP (custom polyclonal, a gift from G. Kops; 1:5000), mouse α-B56γ (clone A-11, sc-374379, Santa Cruz Biotechnology; 1:1000), mouse α-B56α (clone 23, 610615, BD; 1:1000), mouse α-B56δ (clone H-11, sc-271363, Santa Cruz, 1:1000), rabbit α-B56ε (ARP56694-P050, Aviva, 1:1000), mouse α-PPP2CA (clone 1D6, 05–421, Millipore; 1:5000) and rabbit α-PPP2R1A (clone 81G5, #2041, CST; 1:1000), rabbit α-BubR1 (A300-386A, Bethyl; 1:1000), rabbit α-Axin (C76H11, CST; 1:1000), rabbit α-GEF-H1 (155785, Abcam; 1:1000), rabbit α-Kif4a (A301-074A, Bethyl; 1:1000), rabbit α-RepoMan (HPA030049, Sigma; 1:1000) and rabbit α-Actin (A2066, Sigma; 1:5000) and mouse α-alpha-Tubulin (clone B-5-1-2, T5168, Sigma, 1:5000).

Techniques: Binding Assay

Journal: eLife

Article Title: Division of labour between PP2A-B56 isoforms at the centromere and kinetochore

doi: 10.7554/eLife.42619

Figure Lengend Snippet: ( A and B ) Representative images ( A ) and line plots ( B ) of nocodazole-arrested Flp-in HeLa cells expressing YFP-B56 (B56α, B56β, B56γ1, B56γ3, B56δ and B56ε). For line plots, five kinetochore pairs were analysed per cell, for a total of 10 cells per experiment. Graphs represent the mean intensities (±SD) from 3 independent experiments. Intensity is normalized to the maximum signal in each channel in each experiment. ( C–G ) Flp-in HeLa cells treated with siRNA against B56pool, all B56 isoforms except B56α, or all B56 isoforms except B56γ, were analysed for sister chromatid cohesion, SAC strength, Knl1-MELT dephosphorylation and chromosomal alignment. ( C ) Quantification of percentage of chromosome spreads that contain at least one split centromere. Graph represents mean data (+SD) from 3 independent experiments with 50 metaphase spreads quantified per condition per experiment. ( D ) Time-lapse analysis of cells entering mitosis in the presence of nocodazole and 2.5 µM AZ-3146. The graph represents the cumulative data from 50 cells, which is representative of 3 independent experiments. Representative images ( E ) and quantification ( F ) of relative kinetochore intensities of Knl1-pMELT in cells arrested in prometaphase with nocodazole and treated with MG132 for 30 min, followed by 2.5 µM AZ-3146 for the indicated amount of time. 10 cells were quantified per experiment and the graph displays the mean (+SD) of 3 independent experiments. The individual data points for each experiment can be found in the source data. ( G ) Quantification of chromosome misalignment in cells arrested in metaphase with MG-132. At least 100 cells were scored per condition per experiment and graph represents the mean (-SD) of 3 independent experiments. Misalignments were score as mild (1 to 2 misaligned chromosomes), intermediate (3 to 5 misaligned chromosomes), and severe (>5 misaligned chromosomes). Asterisks indicate significance ( : Welch’s t -test, unpaired, : Mann-Whitney test); ns p>0.05, *p≤0.05, **p≤0.01, ****p≤0.0001. Scale bars, 5µm.

Article Snippet: The following antibodies were used for western blotting (at the final concentration indicated): rabbit α-GFP (custom polyclonal, a gift from G. Kops; 1:5000), mouse α-B56γ (clone A-11, sc-374379, Santa Cruz Biotechnology; 1:1000), mouse α-B56α (clone 23, 610615, BD; 1:1000), mouse α-B56δ (clone H-11, sc-271363, Santa Cruz, 1:1000), rabbit α-B56ε (ARP56694-P050, Aviva, 1:1000), mouse α-PPP2CA (clone 1D6, 05–421, Millipore; 1:5000) and rabbit α-PPP2R1A (clone 81G5, #2041, CST; 1:1000), rabbit α-BubR1 (A300-386A, Bethyl; 1:1000), rabbit α-Axin (C76H11, CST; 1:1000), rabbit α-GEF-H1 (155785, Abcam; 1:1000), rabbit α-Kif4a (A301-074A, Bethyl; 1:1000), rabbit α-RepoMan (HPA030049, Sigma; 1:1000) and rabbit α-Actin (A2066, Sigma; 1:5000) and mouse α-alpha-Tubulin (clone B-5-1-2, T5168, Sigma, 1:5000).

Techniques: Expressing, De-Phosphorylation Assay, MANN-WHITNEY

Journal: eLife

Article Title: Division of labour between PP2A-B56 isoforms at the centromere and kinetochore

doi: 10.7554/eLife.42619

Figure Lengend Snippet: Immunoblot of whole cell lysates from nocodazole-arrested HeLa Flp-in cells treated with the indicated siRNA and probed for B56α, B56γ, B56δ, B56ε or tubulin (note B56β was undetectable). Asterix (*) indicate non-specific bands that do not change upon B56 siRNA.

Article Snippet: The following antibodies were used for western blotting (at the final concentration indicated): rabbit α-GFP (custom polyclonal, a gift from G. Kops; 1:5000), mouse α-B56γ (clone A-11, sc-374379, Santa Cruz Biotechnology; 1:1000), mouse α-B56α (clone 23, 610615, BD; 1:1000), mouse α-B56δ (clone H-11, sc-271363, Santa Cruz, 1:1000), rabbit α-B56ε (ARP56694-P050, Aviva, 1:1000), mouse α-PPP2CA (clone 1D6, 05–421, Millipore; 1:5000) and rabbit α-PPP2R1A (clone 81G5, #2041, CST; 1:1000), rabbit α-BubR1 (A300-386A, Bethyl; 1:1000), rabbit α-Axin (C76H11, CST; 1:1000), rabbit α-GEF-H1 (155785, Abcam; 1:1000), rabbit α-Kif4a (A301-074A, Bethyl; 1:1000), rabbit α-RepoMan (HPA030049, Sigma; 1:1000) and rabbit α-Actin (A2066, Sigma; 1:5000) and mouse α-alpha-Tubulin (clone B-5-1-2, T5168, Sigma, 1:5000).

Techniques: Western Blot

Journal: eLife

Article Title: Division of labour between PP2A-B56 isoforms at the centromere and kinetochore

doi: 10.7554/eLife.42619

Figure Lengend Snippet: ( A ) Schematic representation of the strategy used for CRISPR/Cas9 YFP-tagging of B56 isoforms α and γ at the N-terminus. ( B ) Immunoblot of whole cell lysates from nocodazole-arrested HeLa cells with or without YFP-endogenously tagged B56α (enB56α) or B56γ (enB56γ). ( C and D ) Representative images ( C ) and line plots ( D ) of nocodazole-arrested Flp-in HeLa cells expressing endogenously tagged YFP-B56α (enB56α) or YFP-B56γ (enB56γ). For line plots, 5 kinetochore pairs were analysed per cell, for a total of 10 cells per experiment. Graphs represent the mean intensities (±SD) from 3 independent experiments. Intensity is normalized to the maximum signal in each channel in each experiment. Scale bars, 5µm.

Article Snippet: The following antibodies were used for western blotting (at the final concentration indicated): rabbit α-GFP (custom polyclonal, a gift from G. Kops; 1:5000), mouse α-B56γ (clone A-11, sc-374379, Santa Cruz Biotechnology; 1:1000), mouse α-B56α (clone 23, 610615, BD; 1:1000), mouse α-B56δ (clone H-11, sc-271363, Santa Cruz, 1:1000), rabbit α-B56ε (ARP56694-P050, Aviva, 1:1000), mouse α-PPP2CA (clone 1D6, 05–421, Millipore; 1:5000) and rabbit α-PPP2R1A (clone 81G5, #2041, CST; 1:1000), rabbit α-BubR1 (A300-386A, Bethyl; 1:1000), rabbit α-Axin (C76H11, CST; 1:1000), rabbit α-GEF-H1 (155785, Abcam; 1:1000), rabbit α-Kif4a (A301-074A, Bethyl; 1:1000), rabbit α-RepoMan (HPA030049, Sigma; 1:1000) and rabbit α-Actin (A2066, Sigma; 1:5000) and mouse α-alpha-Tubulin (clone B-5-1-2, T5168, Sigma, 1:5000).

Techniques: CRISPR, Western Blot, Expressing

Journal: eLife

Article Title: Division of labour between PP2A-B56 isoforms at the centromere and kinetochore

doi: 10.7554/eLife.42619

Figure Lengend Snippet: ( A-D ) Flp-in HeLa cells treated with siRNA against B56 pool and induced to re-express YFP-B56α or YFP-B56γ were analysed for SAC silencing and chromosomal alignment. Representative images ( A ) and quantification of relative kinetochore intensities ( B ) of Knl1-pMELT in cells arrested in prometaphase with nocodazole and treated with MG132 for 30 min followed by 2.5 μM AZ-3146 for the indicated amount of time. 10 cells were quantified per experiment and graphs represent the mean (+SD) of 4 independent experiments. Scale bars, 5µm. ( C ) Time-lapse analysis of cells entering mitosis in the presence of nocodazole and 2.5 µM AZ-3146. The graph represents the cumulative data from 50 cells, which is representative of 3 independent experiments. ( D ) Quantification of chromosome alignment in cells arrested in metaphase with MG-132. At least 100 cells were scored per condition per experiment and graph represents the mean (-SD) of 3 independent experiments. Misalignments were score as mild (1 to 2 misaligned chromosomes), intermediate (3 to 5 misaligned chromosomes), and severe (>5 misaligned chromosomes). Asterisks indicate significance (Mann-Whitney test); ****p≤0.0001.

Article Snippet: The following antibodies were used for western blotting (at the final concentration indicated): rabbit α-GFP (custom polyclonal, a gift from G. Kops; 1:5000), mouse α-B56γ (clone A-11, sc-374379, Santa Cruz Biotechnology; 1:1000), mouse α-B56α (clone 23, 610615, BD; 1:1000), mouse α-B56δ (clone H-11, sc-271363, Santa Cruz, 1:1000), rabbit α-B56ε (ARP56694-P050, Aviva, 1:1000), mouse α-PPP2CA (clone 1D6, 05–421, Millipore; 1:5000) and rabbit α-PPP2R1A (clone 81G5, #2041, CST; 1:1000), rabbit α-BubR1 (A300-386A, Bethyl; 1:1000), rabbit α-Axin (C76H11, CST; 1:1000), rabbit α-GEF-H1 (155785, Abcam; 1:1000), rabbit α-Kif4a (A301-074A, Bethyl; 1:1000), rabbit α-RepoMan (HPA030049, Sigma; 1:1000) and rabbit α-Actin (A2066, Sigma; 1:5000) and mouse α-alpha-Tubulin (clone B-5-1-2, T5168, Sigma, 1:5000).

Techniques: MANN-WHITNEY

Journal: eLife

Article Title: Division of labour between PP2A-B56 isoforms at the centromere and kinetochore

doi: 10.7554/eLife.42619

Figure Lengend Snippet: ( A-G ) The effect of Sgo1 and/or Sgo2 knockdown on YFP-B56α localisation in Flp-in HeLa cells. Representative images ( A, C, F ) and quantifications ( B, D, G ) of relative kinetochore intensity of B56α in cells arrested in prometaphase with nocodazole after knockdown of Sgo2 ( A, B ), Sgo1 ( C, D ), or Sgo1 +Sgo2 ( F, G ). ( E ) shows line plots of Sgo2 and B56α localisation following Sgo1 knockdown; 5 kinetochore pairs were analysed per cell, for a total of 10 cells per experiment. Graphs represent the mean intensities (±SD) from 3 independent experiments. Intensity is normalized to the maximum signal present in each channel within the endogenous B56α experiment. ( H–M ) Flp-in HeLa cells expressing YFP-B56α or YFP-B56γ were transfected with the CB-Sgo2 ( H–K ) or gChr7 +Cas9 DARPIN ( L, M ) and analysed for B56 recruitment in cells arrested in prometaphase with nocodazole. ( H ), ( L ), and J ). are representative images; I ) and K ) are quantifications of relative centromere/kinetochore intensity of the indicated antigen; and M ) is quantification of intensity of Sgo2 over B56 at the Chr7 locus. For all centromere/kinetochore intensity graphs, each dot represents a cell and 10 cells were quantified per experiment for at least 3 independent experiments. The spread of dots indicates the biological variation between individual cells and the errors bars display the variation between the experimental repeats (displayed as -/+SD of the experimental means). Asterisks indicate significance (Mann-Whitney test); ns p>0.05, *p≤0.05, ****p≤0.0001. Scale bars, 5µm.

Article Snippet: The following antibodies were used for western blotting (at the final concentration indicated): rabbit α-GFP (custom polyclonal, a gift from G. Kops; 1:5000), mouse α-B56γ (clone A-11, sc-374379, Santa Cruz Biotechnology; 1:1000), mouse α-B56α (clone 23, 610615, BD; 1:1000), mouse α-B56δ (clone H-11, sc-271363, Santa Cruz, 1:1000), rabbit α-B56ε (ARP56694-P050, Aviva, 1:1000), mouse α-PPP2CA (clone 1D6, 05–421, Millipore; 1:5000) and rabbit α-PPP2R1A (clone 81G5, #2041, CST; 1:1000), rabbit α-BubR1 (A300-386A, Bethyl; 1:1000), rabbit α-Axin (C76H11, CST; 1:1000), rabbit α-GEF-H1 (155785, Abcam; 1:1000), rabbit α-Kif4a (A301-074A, Bethyl; 1:1000), rabbit α-RepoMan (HPA030049, Sigma; 1:1000) and rabbit α-Actin (A2066, Sigma; 1:5000) and mouse α-alpha-Tubulin (clone B-5-1-2, T5168, Sigma, 1:5000).

Techniques: Knockdown, Expressing, Transfection, MANN-WHITNEY

Journal: eLife

Article Title: Division of labour between PP2A-B56 isoforms at the centromere and kinetochore

doi: 10.7554/eLife.42619

Figure Lengend Snippet: ( A-D ) Flp-in HeLa cells expressing YFP-B56α or YFP-B56γ were transfected with CB-Sgo1 and analysed for B56 recruitment. Representative images ( A and C ) and quantifications ( B and D ) of relative centromere/kinetochore intensity of the indicated antigen in cells arrested in prometaphase with nocodazole. 10 cells were quantified per experiment and graphs represent the mean (±SD) of 4 independent experiments. Asterisks indicate significance (Mann-Whitney test); *****p≤0.0001. Scale bars, 5µm.

Article Snippet: The following antibodies were used for western blotting (at the final concentration indicated): rabbit α-GFP (custom polyclonal, a gift from G. Kops; 1:5000), mouse α-B56γ (clone A-11, sc-374379, Santa Cruz Biotechnology; 1:1000), mouse α-B56α (clone 23, 610615, BD; 1:1000), mouse α-B56δ (clone H-11, sc-271363, Santa Cruz, 1:1000), rabbit α-B56ε (ARP56694-P050, Aviva, 1:1000), mouse α-PPP2CA (clone 1D6, 05–421, Millipore; 1:5000) and rabbit α-PPP2R1A (clone 81G5, #2041, CST; 1:1000), rabbit α-BubR1 (A300-386A, Bethyl; 1:1000), rabbit α-Axin (C76H11, CST; 1:1000), rabbit α-GEF-H1 (155785, Abcam; 1:1000), rabbit α-Kif4a (A301-074A, Bethyl; 1:1000), rabbit α-RepoMan (HPA030049, Sigma; 1:1000) and rabbit α-Actin (A2066, Sigma; 1:5000) and mouse α-alpha-Tubulin (clone B-5-1-2, T5168, Sigma, 1:5000).

Techniques: Expressing, Transfection, MANN-WHITNEY

Journal: eLife

Article Title: Division of labour between PP2A-B56 isoforms at the centromere and kinetochore

doi: 10.7554/eLife.42619

Figure Lengend Snippet: B56γ kinetochore localisation in Flp-in HeLa cells after BubR1 knockdown ( A, B, E ) or mutation of the LxxIxE binding pocket (H187A: C ), ( D, F ) in cells arrested in prometaphase with nocodazole. For each condition, representative images ( A, C ), quantification of relative centromere/kinetochore levels ( B, D ) and line plot analysis ( E, F ) depicts the levels and distribution of the indicated antigens. ( G–J ): representative images ( G, I ) and quantification of relative centromere/kinetochore intensities ( H, J ) YFP-B56γ WT or H187A following Sgo1 knockdown ( G, H ) or mutation of the Sgo1 binding region (ΔSgo1). For all centromere/kinetochore intensity graphs, each dot represents a cell and 10 cells were quantified per experiment from at least 3 independent experiments. The spread of dots indicates the biological variation between individual cells and the errors bars display the variation between experimental repeats (displayed as -/+SD of the experimental means). For the line plot analysis, 5 kinetochore pairs were analysed per cell, for a total of 10 cells per experiment. The graph represents the mean intensities (±SD) from at least 3 independent experiments. Intensity is normalized to the maximum signal in each channel in each experiment. Asterisks indicate significance (Mann-Whitney test); ****p≤0.0001. Scale bars, 5µm.

Article Snippet: The following antibodies were used for western blotting (at the final concentration indicated): rabbit α-GFP (custom polyclonal, a gift from G. Kops; 1:5000), mouse α-B56γ (clone A-11, sc-374379, Santa Cruz Biotechnology; 1:1000), mouse α-B56α (clone 23, 610615, BD; 1:1000), mouse α-B56δ (clone H-11, sc-271363, Santa Cruz, 1:1000), rabbit α-B56ε (ARP56694-P050, Aviva, 1:1000), mouse α-PPP2CA (clone 1D6, 05–421, Millipore; 1:5000) and rabbit α-PPP2R1A (clone 81G5, #2041, CST; 1:1000), rabbit α-BubR1 (A300-386A, Bethyl; 1:1000), rabbit α-Axin (C76H11, CST; 1:1000), rabbit α-GEF-H1 (155785, Abcam; 1:1000), rabbit α-Kif4a (A301-074A, Bethyl; 1:1000), rabbit α-RepoMan (HPA030049, Sigma; 1:1000) and rabbit α-Actin (A2066, Sigma; 1:5000) and mouse α-alpha-Tubulin (clone B-5-1-2, T5168, Sigma, 1:5000).

Techniques: Knockdown, Mutagenesis, Binding Assay, MANN-WHITNEY

Journal: eLife

Article Title: Division of labour between PP2A-B56 isoforms at the centromere and kinetochore

doi: 10.7554/eLife.42619

Figure Lengend Snippet: ( A-H ) Flp-in HeLa cells expressing YFP-B56γ were treated with the indicated siRNA. Representative images ( A, C, E and G ) and quantifications of relative centromere/kinetochore intensity ( B, D, F and H ) of the indicated antigen in cells arrested in prometaphase with nocodazole. On the graphs, each dot represents a cell and 10 cells were quantified per experiment from at least 3 independent experiments. The spread of dots indicates the biological variation between individual cells and the errors bars display the variation between experimental repeats (displayed as -/+SD of the experimental means). Asterisks indicate significance (Mann-Whitney test); ns p>0.05, *p≤0.05, ****p≤0.0001. Scale bars, 5µm.

Article Snippet: The following antibodies were used for western blotting (at the final concentration indicated): rabbit α-GFP (custom polyclonal, a gift from G. Kops; 1:5000), mouse α-B56γ (clone A-11, sc-374379, Santa Cruz Biotechnology; 1:1000), mouse α-B56α (clone 23, 610615, BD; 1:1000), mouse α-B56δ (clone H-11, sc-271363, Santa Cruz, 1:1000), rabbit α-B56ε (ARP56694-P050, Aviva, 1:1000), mouse α-PPP2CA (clone 1D6, 05–421, Millipore; 1:5000) and rabbit α-PPP2R1A (clone 81G5, #2041, CST; 1:1000), rabbit α-BubR1 (A300-386A, Bethyl; 1:1000), rabbit α-Axin (C76H11, CST; 1:1000), rabbit α-GEF-H1 (155785, Abcam; 1:1000), rabbit α-Kif4a (A301-074A, Bethyl; 1:1000), rabbit α-RepoMan (HPA030049, Sigma; 1:1000) and rabbit α-Actin (A2066, Sigma; 1:5000) and mouse α-alpha-Tubulin (clone B-5-1-2, T5168, Sigma, 1:5000).

Techniques: Expressing, MANN-WHITNEY

Journal: eLife

Article Title: Division of labour between PP2A-B56 isoforms at the centromere and kinetochore

doi: 10.7554/eLife.42619

Figure Lengend Snippet: Flp-in HeLa cells expressing YFP-B56γ or YFP-B56γ ΔSgo1 were untransfected or transfected with CB-Sgo1. Representative images ( A, C ) and quantification ( B, D ) of relative kinetochore intensity of the indicated antigen in interphase cells or cells arrested in prometaphase with nocodazole. On the graphs, each dot represents a cell and 10 cells were quantified per experiment from at least three independent experiments. The spread of dots indicates the biological variation between individual cells and the errors bars display the variation between experimental repeats (displayed as -/+SD of the experimental means). Asterisks indicate significance (Mann-Whitney test); ns p>0.05, *p≤0.05, ****p≤0.0001. Scale bars, 5µm.

Article Snippet: The following antibodies were used for western blotting (at the final concentration indicated): rabbit α-GFP (custom polyclonal, a gift from G. Kops; 1:5000), mouse α-B56γ (clone A-11, sc-374379, Santa Cruz Biotechnology; 1:1000), mouse α-B56α (clone 23, 610615, BD; 1:1000), mouse α-B56δ (clone H-11, sc-271363, Santa Cruz, 1:1000), rabbit α-B56ε (ARP56694-P050, Aviva, 1:1000), mouse α-PPP2CA (clone 1D6, 05–421, Millipore; 1:5000) and rabbit α-PPP2R1A (clone 81G5, #2041, CST; 1:1000), rabbit α-BubR1 (A300-386A, Bethyl; 1:1000), rabbit α-Axin (C76H11, CST; 1:1000), rabbit α-GEF-H1 (155785, Abcam; 1:1000), rabbit α-Kif4a (A301-074A, Bethyl; 1:1000), rabbit α-RepoMan (HPA030049, Sigma; 1:1000) and rabbit α-Actin (A2066, Sigma; 1:5000) and mouse α-alpha-Tubulin (clone B-5-1-2, T5168, Sigma, 1:5000).

Techniques: Expressing, Transfection, MANN-WHITNEY

Journal: eLife

Article Title: Division of labour between PP2A-B56 isoforms at the centromere and kinetochore

doi: 10.7554/eLife.42619

Figure Lengend Snippet: ( A ) Immunoblot of the indicated proteins, containing a LxxIxE motif , following YFP immunoprecipitation from nocodazole-arrested Flp-in HeLa cells expressing YFP-B56α or YFP-B56γ. ( B ) Quantification of the mean normalised intensity (+SD) of the indicated antigens in B56α immunoprecipitates, relative to B56γ immunoprecipitates, from at least 3 independent experiments. Representative images ( C ) and line plot analysis ( D ) of YFP-B56α in Flp-in HeLa cells arrested in nocodazole and treated with the indicated siRNA. Each line plot graph represents the mean intensities (±SD) from 3 independent experiments. 5 kinetochore pairs were analysed per cell, for a total of 10 cells per experiment. Intensity is normalized to the maximum signal in each channel in each experiment. Asterisks indicate significance (Welch’s t -test, unpaired); *p≤0.05, **p≤0.01, ****p≤0.0001. Scale bars, 5µm.

Article Snippet: The following antibodies were used for western blotting (at the final concentration indicated): rabbit α-GFP (custom polyclonal, a gift from G. Kops; 1:5000), mouse α-B56γ (clone A-11, sc-374379, Santa Cruz Biotechnology; 1:1000), mouse α-B56α (clone 23, 610615, BD; 1:1000), mouse α-B56δ (clone H-11, sc-271363, Santa Cruz, 1:1000), rabbit α-B56ε (ARP56694-P050, Aviva, 1:1000), mouse α-PPP2CA (clone 1D6, 05–421, Millipore; 1:5000) and rabbit α-PPP2R1A (clone 81G5, #2041, CST; 1:1000), rabbit α-BubR1 (A300-386A, Bethyl; 1:1000), rabbit α-Axin (C76H11, CST; 1:1000), rabbit α-GEF-H1 (155785, Abcam; 1:1000), rabbit α-Kif4a (A301-074A, Bethyl; 1:1000), rabbit α-RepoMan (HPA030049, Sigma; 1:1000) and rabbit α-Actin (A2066, Sigma; 1:5000) and mouse α-alpha-Tubulin (clone B-5-1-2, T5168, Sigma, 1:5000).

Techniques: Western Blot, Immunoprecipitation, Expressing

Journal: eLife

Article Title: Division of labour between PP2A-B56 isoforms at the centromere and kinetochore

doi: 10.7554/eLife.42619

Figure Lengend Snippet: ( A ) Immunoblot of the PP2A subunits following YFP immunoprecipitation from nocodazole-arrested Flp-in HeLa cells expressing YFP-B56α WT or YFP-B56α TKHG. ( B ) Flp-in HeLa cells treated with siRNA against B56 pool and induced to re-express indicted antigens were subjected to time-lapse analysis to determine mitotic timing. The graph represents the cumulative data from 50 cells, which is representative of 3 independent experiments.

Article Snippet: The following antibodies were used for western blotting (at the final concentration indicated): rabbit α-GFP (custom polyclonal, a gift from G. Kops; 1:5000), mouse α-B56γ (clone A-11, sc-374379, Santa Cruz Biotechnology; 1:1000), mouse α-B56α (clone 23, 610615, BD; 1:1000), mouse α-B56δ (clone H-11, sc-271363, Santa Cruz, 1:1000), rabbit α-B56ε (ARP56694-P050, Aviva, 1:1000), mouse α-PPP2CA (clone 1D6, 05–421, Millipore; 1:5000) and rabbit α-PPP2R1A (clone 81G5, #2041, CST; 1:1000), rabbit α-BubR1 (A300-386A, Bethyl; 1:1000), rabbit α-Axin (C76H11, CST; 1:1000), rabbit α-GEF-H1 (155785, Abcam; 1:1000), rabbit α-Kif4a (A301-074A, Bethyl; 1:1000), rabbit α-RepoMan (HPA030049, Sigma; 1:1000) and rabbit α-Actin (A2066, Sigma; 1:5000) and mouse α-alpha-Tubulin (clone B-5-1-2, T5168, Sigma, 1:5000).

Techniques: Western Blot, Immunoprecipitation, Expressing

Journal: eLife

Article Title: Division of labour between PP2A-B56 isoforms at the centromere and kinetochore

doi: 10.7554/eLife.42619

Figure Lengend Snippet: ( A-D ) Flp-in HeLa cells expressing either YFP-B56α WT or TKHG were transfected with the CB-Sgo2 and analysed for B56 recruitment ( A, B ) or gChr7 +dCas9 DARPIN to assess YFP-B56α:Sgo2 co-localisation ( C, D ). Representative images ( A, C ) and quantification of relative kinetochore intensity ( B ) or intensity of Sgo2 over B56α at the Chr7 locus ( D ). For the intensity graphs in B ) and D ), each dot represents a cell and 10 cells were quantified per experiment from at least 3 independent experiments. The spread of dots indicates the biological variation between individual cells and the errors bars display the variation between experimental repeats (displayed as -/+SD of the experimental means). ( E ) Immunoblot of the indicated antigens following immunoprecipitation of YFP from nocodazole-arrested Flp-in HeLa cells expressing YFP- B56γ, YFP-B56α WT or YFP-B56α-TKHG. ( F ) Quantification of the mean normalised intensity (+SD) of indicated antigens in B56α WT or B56α TKHG immunoprecipitates, relative to B56γ, from at least 4 experiments. ( G ) Crystal structure of PP2A-B56γ [accession code 2NPP ] with annotation to indicate the regions that specify localisation to centromeres or kinetochores (see for details of the additional centromere/kinetochore specification regions). Note, the structure is meant only as a guide because the critical regions may be present within B56α, which has not been structurally solved. The B56α WT values are also used in some of the points plotted in . Asterisks indicate significance (Mann-Whitney test, except : Welch’s t -test, unpaired); ns p>0.05, *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001. Scale bars, 5µm.

Article Snippet: The following antibodies were used for western blotting (at the final concentration indicated): rabbit α-GFP (custom polyclonal, a gift from G. Kops; 1:5000), mouse α-B56γ (clone A-11, sc-374379, Santa Cruz Biotechnology; 1:1000), mouse α-B56α (clone 23, 610615, BD; 1:1000), mouse α-B56δ (clone H-11, sc-271363, Santa Cruz, 1:1000), rabbit α-B56ε (ARP56694-P050, Aviva, 1:1000), mouse α-PPP2CA (clone 1D6, 05–421, Millipore; 1:5000) and rabbit α-PPP2R1A (clone 81G5, #2041, CST; 1:1000), rabbit α-BubR1 (A300-386A, Bethyl; 1:1000), rabbit α-Axin (C76H11, CST; 1:1000), rabbit α-GEF-H1 (155785, Abcam; 1:1000), rabbit α-Kif4a (A301-074A, Bethyl; 1:1000), rabbit α-RepoMan (HPA030049, Sigma; 1:1000) and rabbit α-Actin (A2066, Sigma; 1:5000) and mouse α-alpha-Tubulin (clone B-5-1-2, T5168, Sigma, 1:5000).

Techniques: Expressing, Transfection, Western Blot, Immunoprecipitation, MANN-WHITNEY

Journal: eLife

Article Title: Division of labour between PP2A-B56 isoforms at the centromere and kinetochore

doi: 10.7554/eLife.42619

Figure Lengend Snippet: Immunoblot of LxxIxE containing proteins (GEF-H1, BubR1 and RepoMan) following YFP immunoprecipitation from nocodazole-arrested Flp-in HeLa cells expressing YFP-B56α or YFP-B56γ and subjected to control or Sgo2 siRNA, as indicated.

Article Snippet: The following antibodies were used for western blotting (at the final concentration indicated): rabbit α-GFP (custom polyclonal, a gift from G. Kops; 1:5000), mouse α-B56γ (clone A-11, sc-374379, Santa Cruz Biotechnology; 1:1000), mouse α-B56α (clone 23, 610615, BD; 1:1000), mouse α-B56δ (clone H-11, sc-271363, Santa Cruz, 1:1000), rabbit α-B56ε (ARP56694-P050, Aviva, 1:1000), mouse α-PPP2CA (clone 1D6, 05–421, Millipore; 1:5000) and rabbit α-PPP2R1A (clone 81G5, #2041, CST; 1:1000), rabbit α-BubR1 (A300-386A, Bethyl; 1:1000), rabbit α-Axin (C76H11, CST; 1:1000), rabbit α-GEF-H1 (155785, Abcam; 1:1000), rabbit α-Kif4a (A301-074A, Bethyl; 1:1000), rabbit α-RepoMan (HPA030049, Sigma; 1:1000) and rabbit α-Actin (A2066, Sigma; 1:5000) and mouse α-alpha-Tubulin (clone B-5-1-2, T5168, Sigma, 1:5000).

Techniques: Western Blot, Immunoprecipitation, Expressing, Control

Journal: eLife

Article Title: Division of labour between PP2A-B56 isoforms at the centromere and kinetochore

doi: 10.7554/eLife.42619

Figure Lengend Snippet:

Article Snippet: The following antibodies were used for western blotting (at the final concentration indicated): rabbit α-GFP (custom polyclonal, a gift from G. Kops; 1:5000), mouse α-B56γ (clone A-11, sc-374379, Santa Cruz Biotechnology; 1:1000), mouse α-B56α (clone 23, 610615, BD; 1:1000), mouse α-B56δ (clone H-11, sc-271363, Santa Cruz, 1:1000), rabbit α-B56ε (ARP56694-P050, Aviva, 1:1000), mouse α-PPP2CA (clone 1D6, 05–421, Millipore; 1:5000) and rabbit α-PPP2R1A (clone 81G5, #2041, CST; 1:1000), rabbit α-BubR1 (A300-386A, Bethyl; 1:1000), rabbit α-Axin (C76H11, CST; 1:1000), rabbit α-GEF-H1 (155785, Abcam; 1:1000), rabbit α-Kif4a (A301-074A, Bethyl; 1:1000), rabbit α-RepoMan (HPA030049, Sigma; 1:1000) and rabbit α-Actin (A2066, Sigma; 1:5000) and mouse α-alpha-Tubulin (clone B-5-1-2, T5168, Sigma, 1:5000).

Techniques: Recombinant, Clone Assay, Mutagenesis, Plasmid Preparation, Sequencing, Knock-In, Control, Modification, Magnetic Beads, Software, Western Blot, Microscopy

Journal: The Journal of Biological Chemistry

Article Title: Reduction of protein phosphatase 2A (PP2A) complexity reveals cellular functions and dephosphorylation motifs of the PP2A/B′δ holoenzyme

doi: 10.1074/jbc.RA119.011270

Figure Lengend Snippet: Principle of PP2A complexity reduction. A, ribbon diagrams showing heterotrimeric holoenzyme PP2A complexes containing the scaffold (Aα) subunit (green), catalytic (Cα) subunit (pink), and regulatory (Bα, B′γ, and B″β) subunits (turquoise). The zoomed-in panels highlight interacting residues in A (red) and B regulatory (blue) subunits that maintain interactions when swapped. The residue numbers apply to the regulatory subunit family members characterized in this report, Bα, B′δ, and B″α. B, left, PP2A complexity reduction involves complementary charge–swap mutations in Aα and B subunits. B, right, inducible PP2A reduction utilizes Dox-dependent expression of an A/B charge–swap pair together with shRNA-mediated knockdown of endogenous Aα to replace the endogenous PP2A holoenzyme pool (with 4, 5, and 3 regulatory subunits per family) with one defined PP2A holoenzyme. C, schematic of the inducible PP2A reduction vector (see text for details).

Article Snippet: The following commercially-sourced antibodies and labels were used: mouse anti-GFP (clone N86/8, 1:500, Neuromab catalog no. 75-131); rabbit anti-Glu–Glu (1:1000, Cell Signaling catalog no. 2448); mouse anti-PP2A–Bpan (clone 2G9, 1:1000, Upstate catalog no. 05-592);, rat anti-PP2A-A (clone 6G3, 1:1000, Cell Signaling catalog no. 2260); mouse anti-PP2A-Cα (1:1000, BD Biosciences catalog no. 610556); rabbit anti-PP2A/B′δ (1:1000, Abcam catalog no. ab188323); rabbit anti-PP2A-B′ϵ (1:1000, Abcam catalog no. ab1985000); rabbit anti-ERK1/2 (1:500, Santa Cruz Biotechnology catalog no. sc-94); mouse anti-ERK1/2 (1:500, Santa Cruz Biotechnology catalog no. sc-514302); rabbit anti-phospho-ERK1/2 (Thr-202/Tyr-204) (1:1000, Cell Signaling catalog no. 4370); rabbit anti-phospho-PKA substrates (1:1000, Cell Signaling catalog no. 9624); mouse anti-MEK1/2 (1:1000, Cell Signaling catalog no. 4694); rabbit anti-phospho-MEK1/2 (Ser-217/Ser-221) (1:1000, Cell Signaling catalog no. 9154); rabbit anti-phosphoribosomal protein S6 (Ser-235/Ser-236) (1:1000, Cell Signaling catalog no. 4858); rabbit anti-phosphoribosomal protein S6 (Ser-240/Ser-244) (1:1000, Cell Signaling catalog no. 5364); mouse anti-ribosomal protein S6 (1:1000, Cell Signaling catalog no. 2317); goat anti-mouse IRDye® 680RD (LiCOR, catalog no. 926-68070); goat anti-rabbit IRDye® 800CW (LiCOR, catalog no. 926-32211); goat anti-rat IRDye® 800CW (LiCOR, catalog no. 926-32219).

Techniques: Expressing, shRNA, Plasmid Preparation

Journal: The Journal of Biological Chemistry

Article Title: Reduction of protein phosphatase 2A (PP2A) complexity reveals cellular functions and dephosphorylation motifs of the PP2A/B′δ holoenzyme

doi: 10.1074/jbc.RA119.011270

Figure Lengend Snippet: Summary of Aα/B charge–swap mutations tested for complementation Mutations with a check mark (✓) disrupt binding to the wildtype binding partner. A check mark in the complementation (Compl.) column indicates that mutant subunits interact with one another and that mutant Aα does not interact with the wildtype B regulatory subunit, which are the two prerequisites for PP2A complexity reduction. Note Aα R183E impairs endogenous B′δ association, although overexpressed B′δ still binds ( ).

Article Snippet: The following commercially-sourced antibodies and labels were used: mouse anti-GFP (clone N86/8, 1:500, Neuromab catalog no. 75-131); rabbit anti-Glu–Glu (1:1000, Cell Signaling catalog no. 2448); mouse anti-PP2A–Bpan (clone 2G9, 1:1000, Upstate catalog no. 05-592);, rat anti-PP2A-A (clone 6G3, 1:1000, Cell Signaling catalog no. 2260); mouse anti-PP2A-Cα (1:1000, BD Biosciences catalog no. 610556); rabbit anti-PP2A/B′δ (1:1000, Abcam catalog no. ab188323); rabbit anti-PP2A-B′ϵ (1:1000, Abcam catalog no. ab1985000); rabbit anti-ERK1/2 (1:500, Santa Cruz Biotechnology catalog no. sc-94); mouse anti-ERK1/2 (1:500, Santa Cruz Biotechnology catalog no. sc-514302); rabbit anti-phospho-ERK1/2 (Thr-202/Tyr-204) (1:1000, Cell Signaling catalog no. 4370); rabbit anti-phospho-PKA substrates (1:1000, Cell Signaling catalog no. 9624); mouse anti-MEK1/2 (1:1000, Cell Signaling catalog no. 4694); rabbit anti-phospho-MEK1/2 (Ser-217/Ser-221) (1:1000, Cell Signaling catalog no. 9154); rabbit anti-phosphoribosomal protein S6 (Ser-235/Ser-236) (1:1000, Cell Signaling catalog no. 4858); rabbit anti-phosphoribosomal protein S6 (Ser-240/Ser-244) (1:1000, Cell Signaling catalog no. 5364); mouse anti-ribosomal protein S6 (1:1000, Cell Signaling catalog no. 2317); goat anti-mouse IRDye® 680RD (LiCOR, catalog no. 926-68070); goat anti-rabbit IRDye® 800CW (LiCOR, catalog no. 926-32211); goat anti-rat IRDye® 800CW (LiCOR, catalog no. 926-32219).

Techniques: Binding Assay, Mutagenesis

Journal: The Journal of Biological Chemistry

Article Title: Reduction of protein phosphatase 2A (PP2A) complexity reveals cellular functions and dephosphorylation motifs of the PP2A/B′δ holoenzyme

doi: 10.1074/jbc.RA119.011270

Figure Lengend Snippet: Complementation between B′δ E290R and Aα R183E. GFP-tagged B′δ wildtype (WT) or E290R was coexpressed with EE-tagged Aα WT or R183E in COS-1 cells in the indicated combinations at 1:1 plasmid ratios, and PP2A holoenzyme formation was assessed by Aα immunoprecipitation (IP) via the EE epitope tag and immunoblotting for the indicated PP2A subunits. A, Western blottings representative of five independent transfections. Molecular mass marker positions are indicated on the right in kDa. B–E, quantification of transfected B′δ-GFP (B), endogenous pan-B (C), B′δ (D), and B′ϵ (E) in the immunoprecipitate. Signals are normalized to immunoprecipitated Aα-EE levels in the same lane and further normalized to the WT/WT condition. Shown are means ± S.E. as well as individual data points from five independent experiments. Significance levels are indicated as p values determined by one-way ANOVA followed by Dunnett's post hoc test.

Article Snippet: The following commercially-sourced antibodies and labels were used: mouse anti-GFP (clone N86/8, 1:500, Neuromab catalog no. 75-131); rabbit anti-Glu–Glu (1:1000, Cell Signaling catalog no. 2448); mouse anti-PP2A–Bpan (clone 2G9, 1:1000, Upstate catalog no. 05-592);, rat anti-PP2A-A (clone 6G3, 1:1000, Cell Signaling catalog no. 2260); mouse anti-PP2A-Cα (1:1000, BD Biosciences catalog no. 610556); rabbit anti-PP2A/B′δ (1:1000, Abcam catalog no. ab188323); rabbit anti-PP2A-B′ϵ (1:1000, Abcam catalog no. ab1985000); rabbit anti-ERK1/2 (1:500, Santa Cruz Biotechnology catalog no. sc-94); mouse anti-ERK1/2 (1:500, Santa Cruz Biotechnology catalog no. sc-514302); rabbit anti-phospho-ERK1/2 (Thr-202/Tyr-204) (1:1000, Cell Signaling catalog no. 4370); rabbit anti-phospho-PKA substrates (1:1000, Cell Signaling catalog no. 9624); mouse anti-MEK1/2 (1:1000, Cell Signaling catalog no. 4694); rabbit anti-phospho-MEK1/2 (Ser-217/Ser-221) (1:1000, Cell Signaling catalog no. 9154); rabbit anti-phosphoribosomal protein S6 (Ser-235/Ser-236) (1:1000, Cell Signaling catalog no. 4858); rabbit anti-phosphoribosomal protein S6 (Ser-240/Ser-244) (1:1000, Cell Signaling catalog no. 5364); mouse anti-ribosomal protein S6 (1:1000, Cell Signaling catalog no. 2317); goat anti-mouse IRDye® 680RD (LiCOR, catalog no. 926-68070); goat anti-rabbit IRDye® 800CW (LiCOR, catalog no. 926-32211); goat anti-rat IRDye® 800CW (LiCOR, catalog no. 926-32219).

Techniques: Plasmid Preparation, Immunoprecipitation, Western Blot, Transfection, Marker

Journal: The Journal of Biological Chemistry

Article Title: Reduction of protein phosphatase 2A (PP2A) complexity reveals cellular functions and dephosphorylation motifs of the PP2A/B′δ holoenzyme

doi: 10.1074/jbc.RA119.011270

Figure Lengend Snippet: Complementation between Bα RR169EE and Aα EE100RR. GFP-tagged Bα wildtype (WT) or RR169EE was coexpressed with EE-tagged Aα WT or EE100RR in COS-1 cells in the indicated combinations at 1:1 plasmid ratios, and PP2A holoenzyme formation was assessed by Aα immunoprecipitation (IP) via the EE epitope tag and immunoblotting for the indicated PP2A subunits. A, Western blottings representative of four independent experiments. Molecular mass marker positions are indicated on the right in kDa. B–E, quantification of transfected Bα-GFP (B), endogenous B (pan-specific antibody, C), endogenous B′δ (D), and B′ϵ (E) in the immunoprecipitate. Signals are normalized to immunoprecipitated Aα-EE levels in the same lane and further normalized to the WT/WT condition. Shown are means ± S.E. as well as individual data points from four independent experiments. Significance levels are indicated as p values obtained by one-way ANOVA followed by Dunnett's post hoc test.

Article Snippet: The following commercially-sourced antibodies and labels were used: mouse anti-GFP (clone N86/8, 1:500, Neuromab catalog no. 75-131); rabbit anti-Glu–Glu (1:1000, Cell Signaling catalog no. 2448); mouse anti-PP2A–Bpan (clone 2G9, 1:1000, Upstate catalog no. 05-592);, rat anti-PP2A-A (clone 6G3, 1:1000, Cell Signaling catalog no. 2260); mouse anti-PP2A-Cα (1:1000, BD Biosciences catalog no. 610556); rabbit anti-PP2A/B′δ (1:1000, Abcam catalog no. ab188323); rabbit anti-PP2A-B′ϵ (1:1000, Abcam catalog no. ab1985000); rabbit anti-ERK1/2 (1:500, Santa Cruz Biotechnology catalog no. sc-94); mouse anti-ERK1/2 (1:500, Santa Cruz Biotechnology catalog no. sc-514302); rabbit anti-phospho-ERK1/2 (Thr-202/Tyr-204) (1:1000, Cell Signaling catalog no. 4370); rabbit anti-phospho-PKA substrates (1:1000, Cell Signaling catalog no. 9624); mouse anti-MEK1/2 (1:1000, Cell Signaling catalog no. 4694); rabbit anti-phospho-MEK1/2 (Ser-217/Ser-221) (1:1000, Cell Signaling catalog no. 9154); rabbit anti-phosphoribosomal protein S6 (Ser-235/Ser-236) (1:1000, Cell Signaling catalog no. 4858); rabbit anti-phosphoribosomal protein S6 (Ser-240/Ser-244) (1:1000, Cell Signaling catalog no. 5364); mouse anti-ribosomal protein S6 (1:1000, Cell Signaling catalog no. 2317); goat anti-mouse IRDye® 680RD (LiCOR, catalog no. 926-68070); goat anti-rabbit IRDye® 800CW (LiCOR, catalog no. 926-32211); goat anti-rat IRDye® 800CW (LiCOR, catalog no. 926-32219).

Techniques: Plasmid Preparation, Immunoprecipitation, Western Blot, Marker, Transfection

Journal: The Journal of Biological Chemistry

Article Title: Reduction of protein phosphatase 2A (PP2A) complexity reveals cellular functions and dephosphorylation motifs of the PP2A/B′δ holoenzyme

doi: 10.1074/jbc.RA119.011270

Figure Lengend Snippet: Complementation between B″α R361E and Aα E100R. GFP-tagged B″α wildtype (WT) or R361E was coexpressed with EE-tagged Aα WT or E100R at 3:1 plasmid ratios, and PP2A holoenzyme formation was assessed by Aα immunoprecipitation (IP) via the EE epitope tag and immunoblotting for the indicated PP2A subunits. A, Western blottings representative of six independent transfections. Positions of molecular mass markers (in kDa) are indicated on the right. In the B′δ blots, the white line indicates the removal of the irrelevant lanes of the same blots. B–E, quantification of transfected B″α-GFP (B), endogenous pan-B (C), B′δ (D), and B′ϵ (E) in the immunoprecipitate. Signals are normalized to immunoprecipitated Aα-EE levels in the same lane and further normalized to the WT/WT condition. Shown are means ± S.E. as well as individual data points from six independent experiments. Significance levels are indicated as p values determined by one-way ANOVA followed by Dunnett's post hoc test.

Article Snippet: The following commercially-sourced antibodies and labels were used: mouse anti-GFP (clone N86/8, 1:500, Neuromab catalog no. 75-131); rabbit anti-Glu–Glu (1:1000, Cell Signaling catalog no. 2448); mouse anti-PP2A–Bpan (clone 2G9, 1:1000, Upstate catalog no. 05-592);, rat anti-PP2A-A (clone 6G3, 1:1000, Cell Signaling catalog no. 2260); mouse anti-PP2A-Cα (1:1000, BD Biosciences catalog no. 610556); rabbit anti-PP2A/B′δ (1:1000, Abcam catalog no. ab188323); rabbit anti-PP2A-B′ϵ (1:1000, Abcam catalog no. ab1985000); rabbit anti-ERK1/2 (1:500, Santa Cruz Biotechnology catalog no. sc-94); mouse anti-ERK1/2 (1:500, Santa Cruz Biotechnology catalog no. sc-514302); rabbit anti-phospho-ERK1/2 (Thr-202/Tyr-204) (1:1000, Cell Signaling catalog no. 4370); rabbit anti-phospho-PKA substrates (1:1000, Cell Signaling catalog no. 9624); mouse anti-MEK1/2 (1:1000, Cell Signaling catalog no. 4694); rabbit anti-phospho-MEK1/2 (Ser-217/Ser-221) (1:1000, Cell Signaling catalog no. 9154); rabbit anti-phosphoribosomal protein S6 (Ser-235/Ser-236) (1:1000, Cell Signaling catalog no. 4858); rabbit anti-phosphoribosomal protein S6 (Ser-240/Ser-244) (1:1000, Cell Signaling catalog no. 5364); mouse anti-ribosomal protein S6 (1:1000, Cell Signaling catalog no. 2317); goat anti-mouse IRDye® 680RD (LiCOR, catalog no. 926-68070); goat anti-rabbit IRDye® 800CW (LiCOR, catalog no. 926-32211); goat anti-rat IRDye® 800CW (LiCOR, catalog no. 926-32219).

Techniques: Plasmid Preparation, Immunoprecipitation, Western Blot, Transfection

Journal: The Journal of Biological Chemistry

Article Title: Reduction of protein phosphatase 2A (PP2A) complexity reveals cellular functions and dephosphorylation motifs of the PP2A/B′δ holoenzyme

doi: 10.1074/jbc.RA119.011270

Figure Lengend Snippet: Complementation between B″ K251D,K331D and Aα D61K,D177K. Wildtype (WT) or mutant B″α-GFP was coexpressed with WT or mutant Aα-EE at 3:1 plasmid ratios, and PP2A holoenzyme assembly was assessed by Aα-EE immunoprecipitation (IP) and immunoblotting for the indicated PP2A subunits. A, Western blottings representative of four independent transfections. Positions of molecular mass markers (in kDa) are indicated on the right. B–E, quantification of transfected B″α-GFP (B), endogenous pan-B (C), B′δ (D), and B′ϵ (E) in the immunoprecipitate. Signals are normalized to immunoprecipitated Aα-EE levels in the same lane and to the WT/WT condition (means ± S.E. and individual data points from four transfections). Significance levels are indicated as p values determined by one-way ANOVA followed by Dunnett's post hoc test.

Article Snippet: The following commercially-sourced antibodies and labels were used: mouse anti-GFP (clone N86/8, 1:500, Neuromab catalog no. 75-131); rabbit anti-Glu–Glu (1:1000, Cell Signaling catalog no. 2448); mouse anti-PP2A–Bpan (clone 2G9, 1:1000, Upstate catalog no. 05-592);, rat anti-PP2A-A (clone 6G3, 1:1000, Cell Signaling catalog no. 2260); mouse anti-PP2A-Cα (1:1000, BD Biosciences catalog no. 610556); rabbit anti-PP2A/B′δ (1:1000, Abcam catalog no. ab188323); rabbit anti-PP2A-B′ϵ (1:1000, Abcam catalog no. ab1985000); rabbit anti-ERK1/2 (1:500, Santa Cruz Biotechnology catalog no. sc-94); mouse anti-ERK1/2 (1:500, Santa Cruz Biotechnology catalog no. sc-514302); rabbit anti-phospho-ERK1/2 (Thr-202/Tyr-204) (1:1000, Cell Signaling catalog no. 4370); rabbit anti-phospho-PKA substrates (1:1000, Cell Signaling catalog no. 9624); mouse anti-MEK1/2 (1:1000, Cell Signaling catalog no. 4694); rabbit anti-phospho-MEK1/2 (Ser-217/Ser-221) (1:1000, Cell Signaling catalog no. 9154); rabbit anti-phosphoribosomal protein S6 (Ser-235/Ser-236) (1:1000, Cell Signaling catalog no. 4858); rabbit anti-phosphoribosomal protein S6 (Ser-240/Ser-244) (1:1000, Cell Signaling catalog no. 5364); mouse anti-ribosomal protein S6 (1:1000, Cell Signaling catalog no. 2317); goat anti-mouse IRDye® 680RD (LiCOR, catalog no. 926-68070); goat anti-rabbit IRDye® 800CW (LiCOR, catalog no. 926-32211); goat anti-rat IRDye® 800CW (LiCOR, catalog no. 926-32219).

Techniques: Mutagenesis, Plasmid Preparation, Immunoprecipitation, Western Blot, Transfection

Journal: The Journal of Biological Chemistry

Article Title: Reduction of protein phosphatase 2A (PP2A) complexity reveals cellular functions and dephosphorylation motifs of the PP2A/B′δ holoenzyme

doi: 10.1074/jbc.RA119.011270

Figure Lengend Snippet: Complementation between B″α K251D and Aα D61K. Wildtype (WT) or mutant B″α-GFP was coexpressed with WT or mutant Aα-EE at 3:1 plasmid ratios, and PP2A holoenzyme assembly was assessed by Aα-EE immunoprecipitation (IP) and immunoblotting for the indicated PP2A subunits. A, Western blottings representative of four independent transfections. Positions of molecular mass markers (in kDa) are indicated on the right. B–E, quantification of transfected B″α-GFP (B), endogenous pan-B (C), B′δ (D), and B′ϵ (E) in the immunoprecipitate. Signals are normalized to immunoprecipitated Aα-EE levels in the same lane and to the WT/WT condition (means ± S.E. and individual data points from four transfections). Significance levels are indicated as p values determined by one-way ANOVA followed by Dunnett's post hoc test.

Article Snippet: The following commercially-sourced antibodies and labels were used: mouse anti-GFP (clone N86/8, 1:500, Neuromab catalog no. 75-131); rabbit anti-Glu–Glu (1:1000, Cell Signaling catalog no. 2448); mouse anti-PP2A–Bpan (clone 2G9, 1:1000, Upstate catalog no. 05-592);, rat anti-PP2A-A (clone 6G3, 1:1000, Cell Signaling catalog no. 2260); mouse anti-PP2A-Cα (1:1000, BD Biosciences catalog no. 610556); rabbit anti-PP2A/B′δ (1:1000, Abcam catalog no. ab188323); rabbit anti-PP2A-B′ϵ (1:1000, Abcam catalog no. ab1985000); rabbit anti-ERK1/2 (1:500, Santa Cruz Biotechnology catalog no. sc-94); mouse anti-ERK1/2 (1:500, Santa Cruz Biotechnology catalog no. sc-514302); rabbit anti-phospho-ERK1/2 (Thr-202/Tyr-204) (1:1000, Cell Signaling catalog no. 4370); rabbit anti-phospho-PKA substrates (1:1000, Cell Signaling catalog no. 9624); mouse anti-MEK1/2 (1:1000, Cell Signaling catalog no. 4694); rabbit anti-phospho-MEK1/2 (Ser-217/Ser-221) (1:1000, Cell Signaling catalog no. 9154); rabbit anti-phosphoribosomal protein S6 (Ser-235/Ser-236) (1:1000, Cell Signaling catalog no. 4858); rabbit anti-phosphoribosomal protein S6 (Ser-240/Ser-244) (1:1000, Cell Signaling catalog no. 5364); mouse anti-ribosomal protein S6 (1:1000, Cell Signaling catalog no. 2317); goat anti-mouse IRDye® 680RD (LiCOR, catalog no. 926-68070); goat anti-rabbit IRDye® 800CW (LiCOR, catalog no. 926-32211); goat anti-rat IRDye® 800CW (LiCOR, catalog no. 926-32219).

Techniques: Mutagenesis, Plasmid Preparation, Immunoprecipitation, Western Blot, Transfection

Journal: The Journal of Biological Chemistry

Article Title: Reduction of protein phosphatase 2A (PP2A) complexity reveals cellular functions and dephosphorylation motifs of the PP2A/B′δ holoenzyme

doi: 10.1074/jbc.RA119.011270

Figure Lengend Snippet: Comparison of PP2A subunit expression in inducible PP2A/B′δ RE versus OE cell lines. Clonal HEK293 cell lines with either inducible PP2A/B′δ RE (Aα R183E-B′δ E290R) or OE were treated for the indicated number of days with Dox before immunoblotting total lysates for the indicated proteins. A, Western blottings from one representative experiment. Positions of molecular mass markers (in kDa) are indicated on the right. B–D, quantification of endogenous B (pan-specific antibody, B), B′δ (C), and B′ϵ levels (D) normalized to ERK and day 0. Data shown are means ± S.E. of four (RE) and eight (OE) clonal cell lines. Individual symbols are means of two to three replicate analyses of each cell clone. Listed p values are based on one-way ANOVA followed by Dunnett's post hoc test.

Article Snippet: The following commercially-sourced antibodies and labels were used: mouse anti-GFP (clone N86/8, 1:500, Neuromab catalog no. 75-131); rabbit anti-Glu–Glu (1:1000, Cell Signaling catalog no. 2448); mouse anti-PP2A–Bpan (clone 2G9, 1:1000, Upstate catalog no. 05-592);, rat anti-PP2A-A (clone 6G3, 1:1000, Cell Signaling catalog no. 2260); mouse anti-PP2A-Cα (1:1000, BD Biosciences catalog no. 610556); rabbit anti-PP2A/B′δ (1:1000, Abcam catalog no. ab188323); rabbit anti-PP2A-B′ϵ (1:1000, Abcam catalog no. ab1985000); rabbit anti-ERK1/2 (1:500, Santa Cruz Biotechnology catalog no. sc-94); mouse anti-ERK1/2 (1:500, Santa Cruz Biotechnology catalog no. sc-514302); rabbit anti-phospho-ERK1/2 (Thr-202/Tyr-204) (1:1000, Cell Signaling catalog no. 4370); rabbit anti-phospho-PKA substrates (1:1000, Cell Signaling catalog no. 9624); mouse anti-MEK1/2 (1:1000, Cell Signaling catalog no. 4694); rabbit anti-phospho-MEK1/2 (Ser-217/Ser-221) (1:1000, Cell Signaling catalog no. 9154); rabbit anti-phosphoribosomal protein S6 (Ser-235/Ser-236) (1:1000, Cell Signaling catalog no. 4858); rabbit anti-phosphoribosomal protein S6 (Ser-240/Ser-244) (1:1000, Cell Signaling catalog no. 5364); mouse anti-ribosomal protein S6 (1:1000, Cell Signaling catalog no. 2317); goat anti-mouse IRDye® 680RD (LiCOR, catalog no. 926-68070); goat anti-rabbit IRDye® 800CW (LiCOR, catalog no. 926-32211); goat anti-rat IRDye® 800CW (LiCOR, catalog no. 926-32219).

Techniques: Expressing, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Reduction of protein phosphatase 2A (PP2A) complexity reveals cellular functions and dephosphorylation motifs of the PP2A/B′δ holoenzyme

doi: 10.1074/jbc.RA119.011270

Figure Lengend Snippet: PP2A complexity reduction, but not overexpression, detects B′δ-mediated dephosphorylation events. PP2A/B′δ RE and OE cell lines were treated ± doxycycline for 3 days, serum-starved for 4–6 h, and then stimulated with isoproterenol (1 μm, 5 min). β-Adrenergic signaling was terminated by subsequent incubation with propranolol (prop, 10 μm) and H89 (50 μm) for 0–5 min, after which SDS lysates were prepared and analyzed by immunoblotting for the indicated total and phospho (p)-proteins. The top of each panel shows representative blots. Positions of molecular mass markers (in kDa) are indicated on the right. The graphs at the bottom of each panel depict densitometry as means ± S.E. of three clonal cell lines. A and B, pERK to total ERK ratios were normalized to the basal (ba), no Dox condition. C and D, p-PKA substrate signals were first normalized to total protein (Ponceau S stain). Signals of bands labeled I and III were then normalized to the basal, no Dox condition, whereas band II signals were normalized to the no Dox, 5-min time point. Error bars smaller than symbols are not shown. Asterisks denote significant differences between +Dox and −Dox groups based on two-way ANOVA analysis followed by Sidak's multiple comparison post hoc test. *, p < 0.05; **, p < 0.01; and ****, p < 0.0001.

Article Snippet: The following commercially-sourced antibodies and labels were used: mouse anti-GFP (clone N86/8, 1:500, Neuromab catalog no. 75-131); rabbit anti-Glu–Glu (1:1000, Cell Signaling catalog no. 2448); mouse anti-PP2A–Bpan (clone 2G9, 1:1000, Upstate catalog no. 05-592);, rat anti-PP2A-A (clone 6G3, 1:1000, Cell Signaling catalog no. 2260); mouse anti-PP2A-Cα (1:1000, BD Biosciences catalog no. 610556); rabbit anti-PP2A/B′δ (1:1000, Abcam catalog no. ab188323); rabbit anti-PP2A-B′ϵ (1:1000, Abcam catalog no. ab1985000); rabbit anti-ERK1/2 (1:500, Santa Cruz Biotechnology catalog no. sc-94); mouse anti-ERK1/2 (1:500, Santa Cruz Biotechnology catalog no. sc-514302); rabbit anti-phospho-ERK1/2 (Thr-202/Tyr-204) (1:1000, Cell Signaling catalog no. 4370); rabbit anti-phospho-PKA substrates (1:1000, Cell Signaling catalog no. 9624); mouse anti-MEK1/2 (1:1000, Cell Signaling catalog no. 4694); rabbit anti-phospho-MEK1/2 (Ser-217/Ser-221) (1:1000, Cell Signaling catalog no. 9154); rabbit anti-phosphoribosomal protein S6 (Ser-235/Ser-236) (1:1000, Cell Signaling catalog no. 4858); rabbit anti-phosphoribosomal protein S6 (Ser-240/Ser-244) (1:1000, Cell Signaling catalog no. 5364); mouse anti-ribosomal protein S6 (1:1000, Cell Signaling catalog no. 2317); goat anti-mouse IRDye® 680RD (LiCOR, catalog no. 926-68070); goat anti-rabbit IRDye® 800CW (LiCOR, catalog no. 926-32211); goat anti-rat IRDye® 800CW (LiCOR, catalog no. 926-32219).

Techniques: Over Expression, De-Phosphorylation Assay, Incubation, Western Blot, Staining, Labeling

Journal: The Journal of Biological Chemistry

Article Title: Reduction of protein phosphatase 2A (PP2A) complexity reveals cellular functions and dephosphorylation motifs of the PP2A/B′δ holoenzyme

doi: 10.1074/jbc.RA119.011270

Figure Lengend Snippet: PP2A complexity reduction identifies putative B′δ substrates in the global phosphoproteome. Three clonal PP2A/B′δ RE and OE cell lines were pooled and treated ± Dox for 3 days, serum-starved for 4–6 h, and then stimulated with isoproterenol (1 μm, 5 min). Urea lysates were digested with trypsin, TMT-labeled, phospho-enriched, and subjected to quantitative LC-MS/MS analysis. After normalization to protein abundance, phosphorylation changes were calculated as the ratio of +Dox to −Dox phosphopeptide levels. A, numbers of phospho-Ser, phospho-Thr, and phospho-Tyr–containing peptides that were at least 1.5-fold depleted by PP2A/B′δ RE and OE. B and C, Venn diagrams illustrating numbers of phosphopeptides (B) and proteins (C) that were either uniquely depleted by RE and OE (numbers to the left and right) or depleted by both RE and OE (numbers in the intersections, see also supporting Excel File S2). Proteins were counted as potential substrates if at least one phosphopeptide was depleted ≥1.5-fold. Because some proteins were identified as phosphopeptides only (not in the total peptide pool), phosphopeptide ratios were calculated without normalization to total protein for C only. D, STRING network analysis (18) shows four major subnetworks and potential protein–protein interactions among the 238 potential PP2A/B′δ RE substrates. Node colors represent different subnetworks based on k means clustering of 5. Substrates that are not connected within the network are not shown.

Article Snippet: The following commercially-sourced antibodies and labels were used: mouse anti-GFP (clone N86/8, 1:500, Neuromab catalog no. 75-131); rabbit anti-Glu–Glu (1:1000, Cell Signaling catalog no. 2448); mouse anti-PP2A–Bpan (clone 2G9, 1:1000, Upstate catalog no. 05-592);, rat anti-PP2A-A (clone 6G3, 1:1000, Cell Signaling catalog no. 2260); mouse anti-PP2A-Cα (1:1000, BD Biosciences catalog no. 610556); rabbit anti-PP2A/B′δ (1:1000, Abcam catalog no. ab188323); rabbit anti-PP2A-B′ϵ (1:1000, Abcam catalog no. ab1985000); rabbit anti-ERK1/2 (1:500, Santa Cruz Biotechnology catalog no. sc-94); mouse anti-ERK1/2 (1:500, Santa Cruz Biotechnology catalog no. sc-514302); rabbit anti-phospho-ERK1/2 (Thr-202/Tyr-204) (1:1000, Cell Signaling catalog no. 4370); rabbit anti-phospho-PKA substrates (1:1000, Cell Signaling catalog no. 9624); mouse anti-MEK1/2 (1:1000, Cell Signaling catalog no. 4694); rabbit anti-phospho-MEK1/2 (Ser-217/Ser-221) (1:1000, Cell Signaling catalog no. 9154); rabbit anti-phosphoribosomal protein S6 (Ser-235/Ser-236) (1:1000, Cell Signaling catalog no. 4858); rabbit anti-phosphoribosomal protein S6 (Ser-240/Ser-244) (1:1000, Cell Signaling catalog no. 5364); mouse anti-ribosomal protein S6 (1:1000, Cell Signaling catalog no. 2317); goat anti-mouse IRDye® 680RD (LiCOR, catalog no. 926-68070); goat anti-rabbit IRDye® 800CW (LiCOR, catalog no. 926-32211); goat anti-rat IRDye® 800CW (LiCOR, catalog no. 926-32219).

Techniques: Labeling, Liquid Chromatography with Mass Spectroscopy

Journal: The Journal of Biological Chemistry

Article Title: Reduction of protein phosphatase 2A (PP2A) complexity reveals cellular functions and dephosphorylation motifs of the PP2A/B′δ holoenzyme

doi: 10.1074/jbc.RA119.011270

Figure Lengend Snippet: RPS6 is a putative substrate of PKA-activated PP2A/B′δ. P2A/B′δ RE cells were treated ± Dox for 3 days, serum-starved for 4–6 h, and then stimulated ± isoproterenol (1 μm, 5 min). SDS lysates were analyzed by PAGE and immunoblotting for total and phospho (p)-ribosomal protein S6 (RPS6; A, pSer-235/236; B, pSer-240/244). The top of each panel shows representative blots with molecular mass markers (in kDa) indicated on the right. The graphs at the bottom of each panel depict pRPS6 to total RPS6 ratios of four clonal cells lines normalized to the basal/−Dox condition as individual data points and as means ± S.E. p values were obtained by one-way ANOVA with Tukey's post hoc test.

Article Snippet: The following commercially-sourced antibodies and labels were used: mouse anti-GFP (clone N86/8, 1:500, Neuromab catalog no. 75-131); rabbit anti-Glu–Glu (1:1000, Cell Signaling catalog no. 2448); mouse anti-PP2A–Bpan (clone 2G9, 1:1000, Upstate catalog no. 05-592);, rat anti-PP2A-A (clone 6G3, 1:1000, Cell Signaling catalog no. 2260); mouse anti-PP2A-Cα (1:1000, BD Biosciences catalog no. 610556); rabbit anti-PP2A/B′δ (1:1000, Abcam catalog no. ab188323); rabbit anti-PP2A-B′ϵ (1:1000, Abcam catalog no. ab1985000); rabbit anti-ERK1/2 (1:500, Santa Cruz Biotechnology catalog no. sc-94); mouse anti-ERK1/2 (1:500, Santa Cruz Biotechnology catalog no. sc-514302); rabbit anti-phospho-ERK1/2 (Thr-202/Tyr-204) (1:1000, Cell Signaling catalog no. 4370); rabbit anti-phospho-PKA substrates (1:1000, Cell Signaling catalog no. 9624); mouse anti-MEK1/2 (1:1000, Cell Signaling catalog no. 4694); rabbit anti-phospho-MEK1/2 (Ser-217/Ser-221) (1:1000, Cell Signaling catalog no. 9154); rabbit anti-phosphoribosomal protein S6 (Ser-235/Ser-236) (1:1000, Cell Signaling catalog no. 4858); rabbit anti-phosphoribosomal protein S6 (Ser-240/Ser-244) (1:1000, Cell Signaling catalog no. 5364); mouse anti-ribosomal protein S6 (1:1000, Cell Signaling catalog no. 2317); goat anti-mouse IRDye® 680RD (LiCOR, catalog no. 926-68070); goat anti-rabbit IRDye® 800CW (LiCOR, catalog no. 926-32211); goat anti-rat IRDye® 800CW (LiCOR, catalog no. 926-32219).

Techniques: Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Reduction of protein phosphatase 2A (PP2A) complexity reveals cellular functions and dephosphorylation motifs of the PP2A/B′δ holoenzyme

doi: 10.1074/jbc.RA119.011270

Figure Lengend Snippet: PP2A/B′δ reduction identifies consensus dephosphorylation motifs in the global phosphoproteome. Phosphopeptides depleted ≥1.5-fold by PP2A/B′δ RE and OE were subjected to motif analysis. A, motif scores (means ± S.E.) of the 45 (RE) and 6 motifs (OE) identified by pLogo (see supporting Excel File S2 for a list of motifs). The p value is based on Student's t test. B and C, motif logos for phospho-Ser- (top) and phospho-Thr–containing peptides (bottom) depleted by PP2A/B′δ RE (B) and OE (C). Pink down arrows indicate positions with significantly enriched residues, which are identified by heights exceeding the significance threshold (red line at +3.75).

Article Snippet: The following commercially-sourced antibodies and labels were used: mouse anti-GFP (clone N86/8, 1:500, Neuromab catalog no. 75-131); rabbit anti-Glu–Glu (1:1000, Cell Signaling catalog no. 2448); mouse anti-PP2A–Bpan (clone 2G9, 1:1000, Upstate catalog no. 05-592);, rat anti-PP2A-A (clone 6G3, 1:1000, Cell Signaling catalog no. 2260); mouse anti-PP2A-Cα (1:1000, BD Biosciences catalog no. 610556); rabbit anti-PP2A/B′δ (1:1000, Abcam catalog no. ab188323); rabbit anti-PP2A-B′ϵ (1:1000, Abcam catalog no. ab1985000); rabbit anti-ERK1/2 (1:500, Santa Cruz Biotechnology catalog no. sc-94); mouse anti-ERK1/2 (1:500, Santa Cruz Biotechnology catalog no. sc-514302); rabbit anti-phospho-ERK1/2 (Thr-202/Tyr-204) (1:1000, Cell Signaling catalog no. 4370); rabbit anti-phospho-PKA substrates (1:1000, Cell Signaling catalog no. 9624); mouse anti-MEK1/2 (1:1000, Cell Signaling catalog no. 4694); rabbit anti-phospho-MEK1/2 (Ser-217/Ser-221) (1:1000, Cell Signaling catalog no. 9154); rabbit anti-phosphoribosomal protein S6 (Ser-235/Ser-236) (1:1000, Cell Signaling catalog no. 4858); rabbit anti-phosphoribosomal protein S6 (Ser-240/Ser-244) (1:1000, Cell Signaling catalog no. 5364); mouse anti-ribosomal protein S6 (1:1000, Cell Signaling catalog no. 2317); goat anti-mouse IRDye® 680RD (LiCOR, catalog no. 926-68070); goat anti-rabbit IRDye® 800CW (LiCOR, catalog no. 926-32211); goat anti-rat IRDye® 800CW (LiCOR, catalog no. 926-32219).

Techniques: De-Phosphorylation Assay

Journal: The EMBO Journal

Article Title: Mechanisms of site‐specific dephosphorylation and kinase opposition imposed by PP2A regulatory subunits

doi: 10.15252/embj.2019103695

Figure Lengend Snippet: A, B Schematic of the B56 inhibitor series and affinities and stoichiometry's for B56α measured by ITC. Global direct fitting shown for one experiment (reverse). Each dot is the integrated heat per injection, and the error bars represent uncertainty with this integrated value. The experiment was done in both direct (B56 in cell) and reverse (B56 in syringe) with similar results. C Time from nuclear envelope breakdown (NEBD) to mitotic exit of cells expressing the indicated B56 inhibitors with each circle representing a single cell. Only cells with similar expression levels of the various B56 inhibitor constructs were analyzed. Median time is indicated by red line. A representative result from at least three independent experiments is shown. At least 25 cells were counted per condition in the experiment shown. A Mann–Whitney U ‐test was used for statistical analysis (ns: non‐significant, *** P ≤ 0.001). D Volcano plot representing mass spectrometry identified proteins co‐purifying with B56 inhibitor versus control inhibitor from HeLa cells. PP2A‐B56 subunits co‐purifying with the B56 inhibitor are indicated. E, F Competition assay in HeLa cells stably expressing RFP‐tagged B56 inhibitor (LxxIxE) or control inhibitor (AxxAxA). YFP‐B56α was transfected into and subsequently purified from these cell lines. Loss of binding of indicated proteins determined by either mass spectrometry (pink—B56 SLiM‐containing protein and known B56 interactor; blue—known B56 interactor, green—B56 SLiM‐containing protein) (E) or Western blotting (F).

Article Snippet: The following antibodies were used at the indicated dilutions: c‐Myc (9E10, sc‐40, 1:1,000, Santa Cruz Biotechnology), Separase (A302‐215A, 1:2,000, Bethyl Laboratories), KIF4A (A301‐074A, 1:1,000, Bethyl Laboratories), Axin1 (#2087, 1:1,000, Cell Signaling Technology), B56α (610615, 1:3,000, BD Biosciences), BubR1 (A300‐995A,1:1,000, Bethyl Laboratories), PP2A catalytic subunit (05‐421, 1:2,000, Millipore), PP2A scaffold subunit (#2041, 1:1,000, Cell Signaling Technology) GFP (ab290, 1:4,000, Abcam), FoxO3 pS413 (#8174, 1:1,000, Cell Signaling Technology), FoxO3 rabbit polyclonal α‐pS253 (Raised against peptide CAPRRRAV(pS)MDNS; 1:500, Moravian Biotechnology), Anti‐RFP (1:1,000; MBL, FM005), Anti‐GFP (1:1,000; Roche, 11814460001), Rabbit anti‐ADAM17 (1:1,000, Abcam, 2051), Rabbit anti‐ADAM17 (1:1,000, Abcam, 39162), Rabbit anti‐EGFR (1:1,000, cell signaling, 2232), Rabbit anti‐pEGFR Y1068 (1:1,000, cell signaling, 2234), Mouse anti‐Transferrin receptor (1:1,000, Invitrogen, 136800), Mouse anti‐GAPDH (1:5,000, Sigma, G8795), rabbit anti‐GFP (1:3,000, Takara Bio Clontech, 632592), Donkey anti‐rabbit‐HRP (1:2,000, GE Healthcare, NA934), Sheep anti‐mouse‐HRP (1:2,000, GE Healthcare, NXA931), and H3pS10 (06‐570, 1:1,000, Millipore).

Techniques: Injection, Expressing, Construct, MANN-WHITNEY, Mass Spectrometry, Control, Competitive Binding Assay, Stable Transfection, Transfection, Purification, Binding Assay, Western Blot

Journal: The EMBO Journal

Article Title: Mechanisms of site‐specific dephosphorylation and kinase opposition imposed by PP2A regulatory subunits

doi: 10.15252/embj.2019103695

Figure Lengend Snippet: Live‐cell imaging of cells expressing doxycycline‐inducible B56 inhibitor. Cell were released from a thymidine block and followed into mitosis. Doxycycline was added at time 0 h. Quantification of mitotic duration of cells from (A) Each circle represents a single cell and median time in mitosis indicated by red line. A representative result from at least three independent experiments is shown. At least 20 cells were counted per condition in the experiment shown. NEBD; nuclear envelope breakdown. Comparison of log ratios of B56‐dependent dephosphorylation sites versus other phosphorylation sites on the same protein. IceLogo representation of over‐ and underrepresented amino acid residues surrounding phosphorylation sites for the down‐regulated phosphorylation sites from experiments presented in Fig A and B. Venn diagram showing overlap of PP2A‐B56‐regulated sites in G1/S and mitosis (M). Schematic of in vitro peptide phosphorylation assay set‐up corresponding to IceLogo of PP2A‐B56‐regulated sites in Fig F. Schematic of LxxIxE inhibition experiment in mitotic lysate corresponding to IceLogo of PP2A‐B56‐regulated sites in Fig G. Mitotic cell lysates treated with thiophosphorylated wt or S62A GST‐Arrp19 for 5 min followed by Western blotting with the antibodies indicated. Source data are available online for this figure.

Article Snippet: The following antibodies were used at the indicated dilutions: c‐Myc (9E10, sc‐40, 1:1,000, Santa Cruz Biotechnology), Separase (A302‐215A, 1:2,000, Bethyl Laboratories), KIF4A (A301‐074A, 1:1,000, Bethyl Laboratories), Axin1 (#2087, 1:1,000, Cell Signaling Technology), B56α (610615, 1:3,000, BD Biosciences), BubR1 (A300‐995A,1:1,000, Bethyl Laboratories), PP2A catalytic subunit (05‐421, 1:2,000, Millipore), PP2A scaffold subunit (#2041, 1:1,000, Cell Signaling Technology) GFP (ab290, 1:4,000, Abcam), FoxO3 pS413 (#8174, 1:1,000, Cell Signaling Technology), FoxO3 rabbit polyclonal α‐pS253 (Raised against peptide CAPRRRAV(pS)MDNS; 1:500, Moravian Biotechnology), Anti‐RFP (1:1,000; MBL, FM005), Anti‐GFP (1:1,000; Roche, 11814460001), Rabbit anti‐ADAM17 (1:1,000, Abcam, 2051), Rabbit anti‐ADAM17 (1:1,000, Abcam, 39162), Rabbit anti‐EGFR (1:1,000, cell signaling, 2232), Rabbit anti‐pEGFR Y1068 (1:1,000, cell signaling, 2234), Mouse anti‐Transferrin receptor (1:1,000, Invitrogen, 136800), Mouse anti‐GAPDH (1:5,000, Sigma, G8795), rabbit anti‐GFP (1:3,000, Takara Bio Clontech, 632592), Donkey anti‐rabbit‐HRP (1:2,000, GE Healthcare, NA934), Sheep anti‐mouse‐HRP (1:2,000, GE Healthcare, NXA931), and H3pS10 (06‐570, 1:1,000, Millipore).

Techniques: Live Cell Imaging, Expressing, Blocking Assay, Comparison, De-Phosphorylation Assay, Phospho-proteomics, In Vitro, Inhibition, Western Blot

Journal: The EMBO Journal

Article Title: Mechanisms of site‐specific dephosphorylation and kinase opposition imposed by PP2A regulatory subunits

doi: 10.15252/embj.2019103695

Figure Lengend Snippet: A Michaelis–Menten kinetic parameters of purified PP2A‐B56α and PP2A‐B55α holoenzymes were determined against the indicated phosphopeptides. Mean and standard deviation shown in plots as black bars ( n = 3 independent experiments). B–D In vitro dephosphorylation by the PP2A‐B55α and PP2A‐B56α holoenzymes of panels of phosphorylated peptides as indicated. Mean and standard deviation shown in plots as black bars ( n = 3 independent experiments).

Article Snippet: The following antibodies were used at the indicated dilutions: c‐Myc (9E10, sc‐40, 1:1,000, Santa Cruz Biotechnology), Separase (A302‐215A, 1:2,000, Bethyl Laboratories), KIF4A (A301‐074A, 1:1,000, Bethyl Laboratories), Axin1 (#2087, 1:1,000, Cell Signaling Technology), B56α (610615, 1:3,000, BD Biosciences), BubR1 (A300‐995A,1:1,000, Bethyl Laboratories), PP2A catalytic subunit (05‐421, 1:2,000, Millipore), PP2A scaffold subunit (#2041, 1:1,000, Cell Signaling Technology) GFP (ab290, 1:4,000, Abcam), FoxO3 pS413 (#8174, 1:1,000, Cell Signaling Technology), FoxO3 rabbit polyclonal α‐pS253 (Raised against peptide CAPRRRAV(pS)MDNS; 1:500, Moravian Biotechnology), Anti‐RFP (1:1,000; MBL, FM005), Anti‐GFP (1:1,000; Roche, 11814460001), Rabbit anti‐ADAM17 (1:1,000, Abcam, 2051), Rabbit anti‐ADAM17 (1:1,000, Abcam, 39162), Rabbit anti‐EGFR (1:1,000, cell signaling, 2232), Rabbit anti‐pEGFR Y1068 (1:1,000, cell signaling, 2234), Mouse anti‐Transferrin receptor (1:1,000, Invitrogen, 136800), Mouse anti‐GAPDH (1:5,000, Sigma, G8795), rabbit anti‐GFP (1:3,000, Takara Bio Clontech, 632592), Donkey anti‐rabbit‐HRP (1:2,000, GE Healthcare, NA934), Sheep anti‐mouse‐HRP (1:2,000, GE Healthcare, NXA931), and H3pS10 (06‐570, 1:1,000, Millipore).

Techniques: Purification, Standard Deviation, In Vitro, De-Phosphorylation Assay

Journal: The EMBO Journal

Article Title: Mechanisms of site‐specific dephosphorylation and kinase opposition imposed by PP2A regulatory subunits

doi: 10.15252/embj.2019103695

Figure Lengend Snippet: A Schematics of engineered FoxO3 constructs. K D measurements of the LxxIxE motifs fused to the C‐terminus of FoxO3 were performed previously (Hertz et al , ; Kruse et al , ). B The indicated YFP‐FoxO3 constructs were transfected into HeLa cells, purified using YFP resin and PP2A‐B56 binding determined by Western blotting. PP2A‐A; scaffold subunit. C, D YFP‐FoxO3 constructs were transfected into HeLa cells, and lysates (pS413) or YFP purifications (pS253) were subjected to Western blotting and probed with the indicated phosphoantibodies. Quantifications arise from five (pS413) or three (pS253) independent experiments. Mean and standard deviation shown in plots as black bars. E Steady‐state localization of the indicated YFP‐FOXO3 variants in HeLa cells. Each data point represents quantification from a single cell. A representative result from at least three independent experiments is shown. At least 25 cells were counted per condition in the experiment shown. Scale bar is 10 μm. Mann–Whitney test significance values; * P < 0.02, *** P < 0.0002. F Dephosphorylation by the PP2A‐B56α holoenzyme complex of substrates with increasing length between phosphorylation sites and binding motifs as depicted. Engineered substrates containing three TP sites were phosphorylated with radioactive ATP using Cdk1 and incubated with the PP2A‐B56α holoenzyme. Removal of radioactive phosphate was monitored over time. Mean and standard deviation from 3 experiments are shown.

Article Snippet: The following antibodies were used at the indicated dilutions: c‐Myc (9E10, sc‐40, 1:1,000, Santa Cruz Biotechnology), Separase (A302‐215A, 1:2,000, Bethyl Laboratories), KIF4A (A301‐074A, 1:1,000, Bethyl Laboratories), Axin1 (#2087, 1:1,000, Cell Signaling Technology), B56α (610615, 1:3,000, BD Biosciences), BubR1 (A300‐995A,1:1,000, Bethyl Laboratories), PP2A catalytic subunit (05‐421, 1:2,000, Millipore), PP2A scaffold subunit (#2041, 1:1,000, Cell Signaling Technology) GFP (ab290, 1:4,000, Abcam), FoxO3 pS413 (#8174, 1:1,000, Cell Signaling Technology), FoxO3 rabbit polyclonal α‐pS253 (Raised against peptide CAPRRRAV(pS)MDNS; 1:500, Moravian Biotechnology), Anti‐RFP (1:1,000; MBL, FM005), Anti‐GFP (1:1,000; Roche, 11814460001), Rabbit anti‐ADAM17 (1:1,000, Abcam, 2051), Rabbit anti‐ADAM17 (1:1,000, Abcam, 39162), Rabbit anti‐EGFR (1:1,000, cell signaling, 2232), Rabbit anti‐pEGFR Y1068 (1:1,000, cell signaling, 2234), Mouse anti‐Transferrin receptor (1:1,000, Invitrogen, 136800), Mouse anti‐GAPDH (1:5,000, Sigma, G8795), rabbit anti‐GFP (1:3,000, Takara Bio Clontech, 632592), Donkey anti‐rabbit‐HRP (1:2,000, GE Healthcare, NA934), Sheep anti‐mouse‐HRP (1:2,000, GE Healthcare, NXA931), and H3pS10 (06‐570, 1:1,000, Millipore).

Techniques: Construct, Transfection, Purification, Binding Assay, Western Blot, Standard Deviation, MANN-WHITNEY, De-Phosphorylation Assay, Phospho-proteomics, Incubation

Journal: The EMBO Journal

Article Title: Mechanisms of site‐specific dephosphorylation and kinase opposition imposed by PP2A regulatory subunits

doi: 10.15252/embj.2019103695

Figure Lengend Snippet: Validation of FoxO3 α‐pS253 antibody. The indicated YFP‐FoxO3 variants were immunopurified and subjected to Western blotting with the indicated antibodies. CIP; calf intestinal phosphatase, 2A; FoxO3 M448A I451A unable to bind PP2A‐B56. Dephosphorylation by the PP2A‐B56α holoenzyme complex of GST‐Cdc20 49–78 substrates with LxxIxE motifs of different binding affinities for B56 as indicated. The mean and standard deviation of 3 independent experiments are shown. Source data are available online for this figure.

Article Snippet: The following antibodies were used at the indicated dilutions: c‐Myc (9E10, sc‐40, 1:1,000, Santa Cruz Biotechnology), Separase (A302‐215A, 1:2,000, Bethyl Laboratories), KIF4A (A301‐074A, 1:1,000, Bethyl Laboratories), Axin1 (#2087, 1:1,000, Cell Signaling Technology), B56α (610615, 1:3,000, BD Biosciences), BubR1 (A300‐995A,1:1,000, Bethyl Laboratories), PP2A catalytic subunit (05‐421, 1:2,000, Millipore), PP2A scaffold subunit (#2041, 1:1,000, Cell Signaling Technology) GFP (ab290, 1:4,000, Abcam), FoxO3 pS413 (#8174, 1:1,000, Cell Signaling Technology), FoxO3 rabbit polyclonal α‐pS253 (Raised against peptide CAPRRRAV(pS)MDNS; 1:500, Moravian Biotechnology), Anti‐RFP (1:1,000; MBL, FM005), Anti‐GFP (1:1,000; Roche, 11814460001), Rabbit anti‐ADAM17 (1:1,000, Abcam, 2051), Rabbit anti‐ADAM17 (1:1,000, Abcam, 39162), Rabbit anti‐EGFR (1:1,000, cell signaling, 2232), Rabbit anti‐pEGFR Y1068 (1:1,000, cell signaling, 2234), Mouse anti‐Transferrin receptor (1:1,000, Invitrogen, 136800), Mouse anti‐GAPDH (1:5,000, Sigma, G8795), rabbit anti‐GFP (1:3,000, Takara Bio Clontech, 632592), Donkey anti‐rabbit‐HRP (1:2,000, GE Healthcare, NA934), Sheep anti‐mouse‐HRP (1:2,000, GE Healthcare, NXA931), and H3pS10 (06‐570, 1:1,000, Millipore).

Techniques: Biomarker Discovery, Western Blot, De-Phosphorylation Assay, Binding Assay, Standard Deviation

Journal: The EMBO Journal

Article Title: Mechanisms of site‐specific dephosphorylation and kinase opposition imposed by PP2A regulatory subunits

doi: 10.15252/embj.2019103695

Figure Lengend Snippet: Domain organization of ADAM17 and conservation of the PP2A‐B56 binding motif. K D values obtained by ITC measurements with full‐length recombinant B56α and indicated ADAM17 variant peptides. The indicated full‐length murine Myc‐ADAM17 derivatives were transfected into HeLa cells stably expressing YFP‐B56α. YFP‐B56α was purified (IP) and ADAM17 binding determined by Western blotting. PP2A‐C; catalytic subunit, PP2A‐A; scaffold subunit. Dephosphorylation by the PP2A‐B56α holoenzyme complex of the indicated phosphorylated GST‐ADAM17 (V724‐C827) fragments. The GST‐ADAM17 (V724‐C827) substrates were phosphorylated with radioactive ATP using protein kinase A and incubated with the PP2A‐B56α holoenzyme. Removal of radioactive phosphate was monitored over time. The mean and standard deviation of 4 independent experiments are shown. Protein expression of ADAM17 variants in the DLD‐1 Adam17 −/− cell line, determined by Western blot. GAPDH was used as an internal loading control. Amphiregulin (AREG) shedding measured by ELISA of conditioned media from untreated, H 2 O 2 treated or irradiated with X‐ray DLD‐1 Adam17 −/− cells (clone #1) expressing full‐length ADAM17 variants (wt, I762A or LEE). Two‐sided, unpaired Student's t ‐test was applied to test for significant differences * P < 0.05, ** P < 0.01, *** P < 0.001. Mean and standard deviation indicated from at least three independent experiments. Exogenous wt and I762A full‐length ADAM17 was immunopurified from Adam17 −/− cells treated with H 2 O 2 and subjected to label‐free LC‐MS/MS to determine differential phosphorylation status of T735 and S808 (S811 in murine ADAM17).

Article Snippet: The following antibodies were used at the indicated dilutions: c‐Myc (9E10, sc‐40, 1:1,000, Santa Cruz Biotechnology), Separase (A302‐215A, 1:2,000, Bethyl Laboratories), KIF4A (A301‐074A, 1:1,000, Bethyl Laboratories), Axin1 (#2087, 1:1,000, Cell Signaling Technology), B56α (610615, 1:3,000, BD Biosciences), BubR1 (A300‐995A,1:1,000, Bethyl Laboratories), PP2A catalytic subunit (05‐421, 1:2,000, Millipore), PP2A scaffold subunit (#2041, 1:1,000, Cell Signaling Technology) GFP (ab290, 1:4,000, Abcam), FoxO3 pS413 (#8174, 1:1,000, Cell Signaling Technology), FoxO3 rabbit polyclonal α‐pS253 (Raised against peptide CAPRRRAV(pS)MDNS; 1:500, Moravian Biotechnology), Anti‐RFP (1:1,000; MBL, FM005), Anti‐GFP (1:1,000; Roche, 11814460001), Rabbit anti‐ADAM17 (1:1,000, Abcam, 2051), Rabbit anti‐ADAM17 (1:1,000, Abcam, 39162), Rabbit anti‐EGFR (1:1,000, cell signaling, 2232), Rabbit anti‐pEGFR Y1068 (1:1,000, cell signaling, 2234), Mouse anti‐Transferrin receptor (1:1,000, Invitrogen, 136800), Mouse anti‐GAPDH (1:5,000, Sigma, G8795), rabbit anti‐GFP (1:3,000, Takara Bio Clontech, 632592), Donkey anti‐rabbit‐HRP (1:2,000, GE Healthcare, NA934), Sheep anti‐mouse‐HRP (1:2,000, GE Healthcare, NXA931), and H3pS10 (06‐570, 1:1,000, Millipore).

Techniques: Binding Assay, Recombinant, Variant Assay, Transfection, Stable Transfection, Expressing, Purification, Western Blot, De-Phosphorylation Assay, Incubation, Standard Deviation, Control, Enzyme-linked Immunosorbent Assay, Irradiation, Liquid Chromatography with Mass Spectroscopy, Phospho-proteomics