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