Journal: PLoS ONE

Article Title: Ferulic Acid Attenuates the Injury-Induced Decrease of Protein Phosphatase 2A Subunit B in Ischemic Brain Injury

doi: 10.1371/journal.pone.0054217

Figure Lengend Snippet: Protein phosphatase 2A (PP2A) subunit B protein spots identified by MALDI-TOF in the cerebral cortices from vehicle+MCAO, ferulic acid+MCAO, vehicle+sham, ferulic acid+sham animals. Animals were treated with vehicle or ferulic acid prior to MCAO. Arrows indicate the protein spots. Mw and pI indicate molecular weight and isoelectrical point, respectively. The intensity of spots was measured using PDQuest software. The ratio of intensity is described as spots intensity of these animals to spots intensity of sham+vehicle animals. Data are shown as mean ± S.E.M. * P <0.05.

Article Snippet: The paraffin sections were washed with PBS, blocked with 1% normal goat serum in PBS for 1 h, and then reacted with anti-PP2A subunit B antibody (diluted 1∶50, Cell Signaling Technology) at 4°C for 15 h. After rinse with PBS, sections were reacted with biotin-conjugated goat anti-rabbit IgG (1∶200 in PBS) for 1 h, followed by incubation with an avidin-biotin-peroxidase complex for 1 h from a Vector ABC Elite kit (Vector Laboratories Inc., Burlingame, CA, USA).

Techniques: Molecular Weight, Software

Journal: PLoS ONE

Article Title: Ferulic Acid Attenuates the Injury-Induced Decrease of Protein Phosphatase 2A Subunit B in Ischemic Brain Injury

doi: 10.1371/journal.pone.0054217

Figure Lengend Snippet: RT-PCR analysis of protein phosphatase 2A (PP2A) subunit B in the cerebral cortices from vehicle+MCAO, ferulic acid+MCAO, vehicle+sham, ferulic acid+sham animals. Animals were treated with vehicle or ferulic acid prior to MCAO. Each lane represents an individual experimental animal. Densitometric analysis is represented as intensity of PP2A subunit B to intensity of actin. Data ( n = 5) are represented as mean ± S.E.M. * P <0.05.

Article Snippet: The paraffin sections were washed with PBS, blocked with 1% normal goat serum in PBS for 1 h, and then reacted with anti-PP2A subunit B antibody (diluted 1∶50, Cell Signaling Technology) at 4°C for 15 h. After rinse with PBS, sections were reacted with biotin-conjugated goat anti-rabbit IgG (1∶200 in PBS) for 1 h, followed by incubation with an avidin-biotin-peroxidase complex for 1 h from a Vector ABC Elite kit (Vector Laboratories Inc., Burlingame, CA, USA).

Techniques: Reverse Transcription Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: Ferulic Acid Attenuates the Injury-Induced Decrease of Protein Phosphatase 2A Subunit B in Ischemic Brain Injury

doi: 10.1371/journal.pone.0054217

Figure Lengend Snippet: Western blot analysis of protein phosphatase 2A (PP2A) subunit B in the cerebral cortices from vehicle+MCAO, ferulic acid+MCAO, vehicle+sham, ferulic acid+sham animals. Animals were treated with vehicle or ferulic acid prior to MCAO. Each lane represents an individual experimental animal. Densitometric analysis is represented as intensity of PP2A subunit B to intensity of actin. Data ( n = 5) are represented as mean ± S.E.M. * P <0.05.

Article Snippet: The paraffin sections were washed with PBS, blocked with 1% normal goat serum in PBS for 1 h, and then reacted with anti-PP2A subunit B antibody (diluted 1∶50, Cell Signaling Technology) at 4°C for 15 h. After rinse with PBS, sections were reacted with biotin-conjugated goat anti-rabbit IgG (1∶200 in PBS) for 1 h, followed by incubation with an avidin-biotin-peroxidase complex for 1 h from a Vector ABC Elite kit (Vector Laboratories Inc., Burlingame, CA, USA).

Techniques: Western Blot

Journal: PLoS ONE

Article Title: Ferulic Acid Attenuates the Injury-Induced Decrease of Protein Phosphatase 2A Subunit B in Ischemic Brain Injury

doi: 10.1371/journal.pone.0054217

Figure Lengend Snippet: Immuno-staining of protein phosphatase 2A (PP2A) subunit B in the cerebral cortices from vehicle+MCAO, ferulic acid+MCAO, vehicle+sham, ferulic acid+sham animals. Animals were treated with vehicle or ferulic acid prior to MCAO. Arrows indicate positive cells of PP2A subunit B. Scale bar = 100 μm.

Article Snippet: The paraffin sections were washed with PBS, blocked with 1% normal goat serum in PBS for 1 h, and then reacted with anti-PP2A subunit B antibody (diluted 1∶50, Cell Signaling Technology) at 4°C for 15 h. After rinse with PBS, sections were reacted with biotin-conjugated goat anti-rabbit IgG (1∶200 in PBS) for 1 h, followed by incubation with an avidin-biotin-peroxidase complex for 1 h from a Vector ABC Elite kit (Vector Laboratories Inc., Burlingame, CA, USA).

Techniques: Immunostaining

Journal: PLoS ONE

Article Title: Ferulic Acid Attenuates the Injury-Induced Decrease of Protein Phosphatase 2A Subunit B in Ischemic Brain Injury

doi: 10.1371/journal.pone.0054217

Figure Lengend Snippet: Cell viability (A) and Western blot analysis of protein phosphatase 2A (PP2A) subunit B (B) in HT22 cells. Glutamate (5 mM) was exposed to HT22 cells for 24 h and ferulic acid (1, 2.5, 5 mM) was treated at 30 min before glutamate exposure. Cellular viability was assessed using the MTT assay (A). Cell survival was expressed as percentage of neuroprotection vs. vehicle set at 100%. Densitometric analysis is represented as intensity of PP2A subunit B to intensity of actin (B). Data ( n = 5) are represented as mean ± S.E.M. * P <0.05.

Article Snippet: The paraffin sections were washed with PBS, blocked with 1% normal goat serum in PBS for 1 h, and then reacted with anti-PP2A subunit B antibody (diluted 1∶50, Cell Signaling Technology) at 4°C for 15 h. After rinse with PBS, sections were reacted with biotin-conjugated goat anti-rabbit IgG (1∶200 in PBS) for 1 h, followed by incubation with an avidin-biotin-peroxidase complex for 1 h from a Vector ABC Elite kit (Vector Laboratories Inc., Burlingame, CA, USA).

Techniques: Western Blot, MTT Assay

Journal: International Journal of Molecular Sciences

Article Title: PP2Ac Modulates AMPK-Mediated Induction of Autophagy in Mycobacterium bovis -Infected Macrophages

doi: 10.3390/ijms20236030

Figure Lengend Snippet: PP2Ac expression in BMDM cells infected with M. bovis . ( A – C ) BMDM cells were infected with M. bovis (multiplicity of infection (MOI) 5, 10, 20 and 40) for 24 h. ( A , B ) Cell lysates were used for PP2Ac protein expression by Western blot (WB) assay. ( C ) Total RNA was extracted and the expression of PP2Acα at mRNA level was determined by quantitative real-time polymerase chain reaction (qRT-PCR). ( D , F ) BMDM cells were infected with M. bovis (MOI 1:10) for indicated time periods. ( D , E ) Cell lysates were used for the expression of PP2Ac protein level by WB. β-actin was used as loading control. ( F ) mRNA level of PP2Acα was determined by qRT-PCR. Data represent the mean ± standard deviation (SD) from three independent experiments. (hpi stands for hours post infection) (* p < 0.05, ** p < 0.01, *** p < 0.001).

Article Snippet: SiRNA-NC (SC-37007), and SiRNA PP2Ac (SC-36302) were purchased from Santa cruz biotechnology (Paso Robles, CA, USA).

Techniques: Expressing, Infection, Western Blot, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Control, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: PP2Ac Modulates AMPK-Mediated Induction of Autophagy in Mycobacterium bovis -Infected Macrophages

doi: 10.3390/ijms20236030

Figure Lengend Snippet: PP2Ac expression in RAW264.7 cells infected with M. bovis . ( A – C ) RAW264.7 cells were infected with M. bovis (MOI 5, 10, 20 and 40) for 24 h. ( A , B ) Cell lysates were used for PP2Ac protein expression by WB assay. ( C ) Total RNA was extracted and the expression PP2Acα at mRNA level was determined by qRT-PCR. ( D , F ) RAW264.7 cells were infected with M. bovis (MOI 1:10) for indicated time period. ( D , E ) Cell lysates were used for the expression of PP2Ac protein by WB. β-actin was used as loading control. ( F ) The mRNA level of PP2Acα was determined by qRT-PCR. Data represent the mean ± SD from three independent experiments. (MOI stands for multiplicity of infection, hpi stands for hours post infection) (* p < 0.05, ** p < 0.01, *** p < 0.001).

Article Snippet: SiRNA-NC (SC-37007), and SiRNA PP2Ac (SC-36302) were purchased from Santa cruz biotechnology (Paso Robles, CA, USA).

Techniques: Expressing, Infection, Quantitative RT-PCR, Control

Journal: International Journal of Molecular Sciences

Article Title: PP2Ac Modulates AMPK-Mediated Induction of Autophagy in Mycobacterium bovis -Infected Macrophages

doi: 10.3390/ijms20236030

Figure Lengend Snippet: Targeting PP2Ac via tyrosine kinase inhibitor (TKI)-nilotinib in M. bovis infected macrophages. ( A – E ) BMDM cells were pretreated with TKI-nilotinib (0 µM or 10 µM) (DMSO 0.1% used as sample diluent control) for 2 h and then infected with M. bovis (MOI 1:10) for indicated time periods. The expression level of ( B ) PP2Ac, ( C ) p -AMPK and ( D ) p -ULK1 were evaluated by using WB and normalized to β-actin. ( E ) The relative expression of PP2Acα at mRNA level was determined by using qRT-PCR. ( F – J ) RAW264.7 cells were treated with TKI-nilotinib (0 µM and 10 µM) followed by M. bovis (MOI 1:10) infection for indicated time periods. The expression levels of ( F ) PP2Ac, ( G ) p -AMPK and ( H ) p -ULK1 cells lysates were determined by WB and normalized to β-actin. ( J ) The relative expression of PP2Acα at mRNA level was determined by using qRT-PCR. Data represent the mean ± SD from three independent experiments. (hpi stands for hours post infection) (* p < 0.05, ** p < 0.01, *** p < 0.001).

Article Snippet: SiRNA-NC (SC-37007), and SiRNA PP2Ac (SC-36302) were purchased from Santa cruz biotechnology (Paso Robles, CA, USA).

Techniques: Infection, Control, Expressing, Quantitative RT-PCR

Journal: International Journal of Molecular Sciences

Article Title: PP2Ac Modulates AMPK-Mediated Induction of Autophagy in Mycobacterium bovis -Infected Macrophages

doi: 10.3390/ijms20236030

Figure Lengend Snippet: Upregulation of PP2Ac abrogates autophagy via modulating AMP-activated protein kinase (AMPK) activation during M. bovis infection. ( A ) BMDM cells were pretreated with okadaic acid (10 nM), TKI-nilotinib (10 µM) alone or okadaic acid + nilotinib followed by M. bovis (MOI 1:10) 24 h (DMSO 0.1% used as solvent control). The expression levels of ( B ) PP2Ac, ( C ) p -AMPK, ( D ) LC3-II, and ( E ) P62 were determined by WB and normalized against β-actin. Data represents the mean ± SD from three independent experiments. (“−“refers the untreated group and “+” refers the treated group for the mentioned treatments ) (* p < 0.05, ** p < 0.01, *** p < 0.001).

Article Snippet: SiRNA-NC (SC-37007), and SiRNA PP2Ac (SC-36302) were purchased from Santa cruz biotechnology (Paso Robles, CA, USA).

Techniques: Activation Assay, Infection, Solvent, Control, Expressing

Journal: International Journal of Molecular Sciences

Article Title: PP2Ac Modulates AMPK-Mediated Induction of Autophagy in Mycobacterium bovis -Infected Macrophages

doi: 10.3390/ijms20236030

Figure Lengend Snippet: Down-regulation of PP2Ac promotes autophagy via mediating AMPK activation during M. bovis infection. ( A ) BMDM cells were pretreated with forskolin (10 µM), TKI-nilotinib (10 µM) alone or forskolin + nilotinib followed by infection with M. bovis (MOI 1:10) 24 h (DMSO 0.1% used as solvent control). The expression levels of ( B ) PP2Ac, ( C ) p -AMPK, ( D ) LC3-II, and ( E ) P62 were determined by WB and normalized against β-actin. Data represents the mean ± SD from three independent experiments. (“−“refers the untreated group and “+” refers the treated group for the mentioned treatments) (* p < 0.05, ** p < 0.01, *** p < 0.001).

Article Snippet: SiRNA-NC (SC-37007), and SiRNA PP2Ac (SC-36302) were purchased from Santa cruz biotechnology (Paso Robles, CA, USA).

Techniques: Activation Assay, Infection, Solvent, Control, Expressing

Journal: International Journal of Molecular Sciences

Article Title: PP2Ac Modulates AMPK-Mediated Induction of Autophagy in Mycobacterium bovis -Infected Macrophages

doi: 10.3390/ijms20236030

Figure Lengend Snippet: PP2Ac promotes intracellular survival of M. bovis in murine macrophages. ( A ) PP2Ac protein level was determined by WB from BMDMs transfected with 50 µM SiRNA negative control and SiRNA PP2Ac for 36 h. ( B ) BMDMs were transfected with 50 µM SiRNA negative control and SiRNA PP2Ac and treated or untreated with TKI-nilotinib (10 µM) followed by infection with M. bovis for 24 h. The expression levels of ( C ) p -AMPK, ( D ) LC3-II and ( E ) P62 were determined by WB and normalized against β-actin. ( F ) The colocalization of LC3 with M. bovis was determined by confocal microscopy. ( G ) The colocalization % of LC3 was calculated by image-J software. ( H ) BMDMs were transfected with 50 µM SiRNA negative control and SiRNA PP2Ac and treated or untreated with nilotinib (10 µM) followed by infection with M. bovis for 24 h. A colony-forming unit (CFU) assay was performed for enumeration of total viable M. bovis bacilli. Data represent the mean ± SD from three independent experiments. (“−“refers the untreated group and “+” refers the treated group for the mentioned treatments ) (* p < 0.05, ** p < 0.01, *** p < 0.001).

Article Snippet: SiRNA-NC (SC-37007), and SiRNA PP2Ac (SC-36302) were purchased from Santa cruz biotechnology (Paso Robles, CA, USA).

Techniques: Transfection, Negative Control, Infection, Expressing, Confocal Microscopy, Software, Colony-forming Unit Assay

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