Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Trimethylamine-N-oxide aggravated the sympathetic excitation in D-galactose induced aging rats by down-regulating P2Y12 receptor in microglia.

doi: 10.1016/j.biopha.2024.116549

Figure Lengend Snippet: Fig. 5. TMAO downregulated P2Y12R in the PVN in the D-gal-induced aging rats. (A) Representative images show immunofluorescence staining of P2Y12R (red) and CD68 (green). Scale bar = 200 µm. (B) Summary data of the percentage of P2Y12R/CD68 positive cells in the PVN of each rat group (n = 3). (C-D) Protein levels of P2Y12R in the PVN detected by Western blot, and (n = 3) for Western blot analysis. Data are presented as the means ± SEM.

Article Snippet: The membranes were incubated with 5% non-fat milk at room temperature and then incubated overnight at 4 °C with the primary antibodies: NLRP3 (1:1000, NBP2–12446, NOUVS, USA), IL-1β (1:1000, NB600–633, NOUVS, USA), p16 (1:1000, ET1608–62, HUABIO, Hangzhou, China), p21 (1:1000, ER1914–57, HUABIO, Hangzhou, China), p53 (1:1000, 21891–1-AP, Proteintech, Wuhan, China), P2Y12R (1:1000, APR-012, Alomone, Israel), and GAPDH (1:10000, 10494–1- AP, Proteintech, Wuhan, China).

Techniques: Immunofluorescence, Staining, Western Blot

Journal: bioRxiv

Article Title: Sodium/Potassium ATPase Alpha 1 Subunit Fine-tunes Platelet GPCR Signaling Function and is Essential for Thrombosis

doi: 10.1101/2024.05.13.593923

Figure Lengend Snippet: A. α 1 +/- and α 1 +/+ platelets in PRP were pooled from five mice from the same strain, and platelet concentration was adjusted to 2.5E+08/mL with PPP. The PRP was divided into 4 equal parts, repleted with MgCl 2 /CaCl 2 to a final concentration of 1 mM, and then treated with 2.5 µM ADP for the indicated periods with rotation. The reaction was stopped by adding an EDTA/PGE1 cocktail containing 500 ng/mL PGE1 and 2 mM EDTA. Platelets were pelleted by a quick spin at 13,000 rpm for 15 seconds, lysed in RIPA buffer, and used for western blot assay of AKT activation. GAPDH was blotted as a loading control. The blot represents two repeats. B. WT platelets pooled from 6 mice were lysed and used for IP of NKA α1 (with ab2872 antibody, 2 µg antibody/1 mg total protein), and then IB for P2Y12 was conducted. IP was also conducted with #1 WT sample pool with normal mouse IgG (2 µg normal IgG/1 mg total protein) as control. C. Platelet lysates prepared from α1 +/- and α 1 +/+ mice were subjected to the Blue-Native PAGE assay. The membrane was blotted for α1 first, stripped, and re-blotted for P2Y12. The image represents two repeats. D. COS-7 cells were transiently transfected with P2Y12-tango plasmid (p-hP2Y12, addgene, #66471), or pEYFP-N1-A2BR (p-hA2BR, addgene, #37202) for 36 h and then cell lysates were used for IP α1 (with ab2872) and immunoblotted for P2Y12. The membrane was stripped and re-blotted for α1 and A2BR (no band was detected in the IP and data was not shown).

Article Snippet: Antibodies to P2Y12 (AGP-098) and A2BR (AAR-003) were purchased from Alomone Lab. Antibodies to NKAα1 subunit (ab2872, ab7671, and ab76020) and P2Y12 (ab184411, ab183066) were purchased from abcam (Waltham, MA).

Techniques: Concentration Assay, Western Blot, Activation Assay, Control, Blue Native PAGE, Membrane, Transfection, Plasmid Preparation

Journal: bioRxiv

Article Title: Sodium/Potassium ATPase Alpha 1 Subunit Fine-tunes Platelet GPCR Signaling Function and is Essential for Thrombosis

doi: 10.1101/2024.05.13.593923

Figure Lengend Snippet: A. COS-7 cells were transiently transfected with plasmids encoding mouse NKA α1 and WT or LGL>SFT mutated mouse P2Y12 for 36 hours and then cells were lysed and used for the IP-IB assay. B & C. Human platelet lysates prepared from two individuals were used for IP of α1 (with ab2872) and then IB for PAR4 and TP. The membranes were stripped and reblotted for α1 (ab76020). HRP-conjugated anti-rabbit IgG light chain antibody was used for the detection of PAR4, TP, and α1 (middle blot) signals. Due to the weak signal of α1 in the IP, the membranes were re-blotted using the HRP-conjugated anti-rabbit whole IgG, and then the α1 signal was visualized by DAB staining. D. COS-7 cells were transiently transfected with plasmids encoding human P2Y12 for 24 hours. The cells were then treated with LGL at the indicated concentrations for an additional 16 hours. Cells were lysed and used for the co-IP assay. GAPDH was blotted for loading control of input. E. COS-7 cells were transiently transfected with plasmids encoding mouse α1 and P2Y12, either WT or LGL(LGI)>SFT mutated genes, in different combinations and then cells were lysed and used for the IP-IB assay. Two dishes of cells transfected with mouse WT α1 and P2Y12 were treated with 100 µM Ouabain or 2 µM Digoxin, respectively, for 1 hour before being harvested for IP-IB assay. SB indicates sample buffer only was loaded in the corresponding lane.

Article Snippet: Antibodies to P2Y12 (AGP-098) and A2BR (AAR-003) were purchased from Alomone Lab. Antibodies to NKAα1 subunit (ab2872, ab7671, and ab76020) and P2Y12 (ab184411, ab183066) were purchased from abcam (Waltham, MA).

Techniques: Transfection, Staining, Co-Immunoprecipitation Assay, Control

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Figure Lengend Snippet: Primary antibodies employed in the study.

Article Snippet: P2Y 12 receptor-ATTO-594 ( intra fl ) , Polyclonal , 125–142 , P2Y 12 receptor , 1 : 50 , Alomone.

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Figure Lengend Snippet: P2Y 12 antibodies validation and RT-PCR analysis. (a) Scheme of human P2RY 12 gene location, transcript variants [ , ], and protein structure, with amino acid epitopes recognized by the used antibodies and highlighted in color ( intra1 , intra2 , and intra fl , red circle; c-ter , green oval). Species conservation for each epitope was calculated by using BLAT tool of UCSC genome browser . (b) Total protein extracts from SH-SY5Y or HEK293 cells expressing Myc-tagged P2Y 12 receptor were subjected to Western blot analysis with the indicated antibodies. (c) Total protein extracted from human, rat and mouse brain, from primary mouse microglia (mMG) and rat oligodendrocyte (OL) cultures were subjected to Western blot analysis with the indicated antibodies. For intra2 antibodies, lots AN01/02/04/0502/0602 were used. (d) RT-PCR using primers specific for P2Y 12 mRNA was performed on total RNA from rat microglia (rMG) and OL. Control lanes show RT-PCR performed without reverse transcriptase enzyme.

Article Snippet: P2Y 12 receptor-ATTO-594 ( intra fl ) , Polyclonal , 125–142 , P2Y 12 receptor , 1 : 50 , Alomone.

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot

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Figure Lengend Snippet: P2Y 12 receptor in dissociated and organotypic primary cultures. (a) Mouse primary cortical microglia were subjected to immunofluorescence and confocal analysis with phalloidin (green, merged field) and P2Y 12 receptor antibodies (red, insets and merged) and Hoechst (white, merged). Scale bars in insets: 20 μ m. (b) Double immunofluorescence and confocal analysis of primary rat mature (OL) and precursor (OPC) oligodendrocytes was performed with antibodies for P2Y 12 receptor, MBP, NG2 . For intra2 antibodies, lots AN01/02/04/0502/0602 were used. (c) Rat cerebellar organotypic cultures were analyzed by double immunofluorescence and confocal microscopy for intra1 (red) and c-ter (red), highlighting different structures (see also insets), and MBP (green).

Article Snippet: P2Y 12 receptor-ATTO-594 ( intra fl ) , Polyclonal , 125–142 , P2Y 12 receptor , 1 : 50 , Alomone.

Techniques: Immunofluorescence, Confocal Microscopy

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Figure Lengend Snippet: P2Y 12 receptor in rat brain tissue. Double immunofluorescence and confocal analysis was performed on sections from rat cerebellum (panels (a), (b), (c), (g), (h), and (i)) and striatum (panels (d), (e), (f), (j), (k), and (l)) with intra1 , intra2 -lots AN01/02/04, intra fl , c-ter (all red), and GFAP (green, inset b1), CD11b (green, insets c1, f1, h1; yellow merged, inset i1; green, panel (k); merged, panel (l)), MBP (yellow merged, inset c2; green, inset e1; green, panels (h) and (i); green, inset j1) antibodies.

Article Snippet: P2Y 12 receptor-ATTO-594 ( intra fl ) , Polyclonal , 125–142 , P2Y 12 receptor , 1 : 50 , Alomone.

Techniques: Immunofluorescence

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Figure Lengend Snippet: Temporal and regional pattern of P2Y 12 expression in SOD1-G93A ALS spinal microglia. (a) Double immunofluorescence and confocal analysis on lumbar spinal cord sections (L3–L5) of wild-type (WT) mice was performed with c-ter antibody (green and yellow, merged and insets), CD11b (left panel, yellow, merged and inset), and CD68 (right panel, red, merged and inset), in both dorsal (DH) and ventral (VH) horns of spinal cord. (b) Double immunofluorescence and confocal analysis on SOD1-G93A lumbar spinal cord sections (L3–L5) at two different stages of ALS disease, that is, 20 weeks, and end stage, was performed with c-ter (green) and CD68 (red) antibodies. (c) Equal amount of total lumbar spinal cord lysates (L3–L5) from WT and SOD1-G93A ( n = 4 for each group) were subjected to Western blotting and immunoreactions with c-ter and CD68 antibodies; anti- β -actin was used for protein normalization. Data represent means ± SEM. Statistical significance was calculated by Student's t -test, * P < 0.05.

Article Snippet: P2Y 12 receptor-ATTO-594 ( intra fl ) , Polyclonal , 125–142 , P2Y 12 receptor , 1 : 50 , Alomone.

Techniques: Expressing, Immunofluorescence, Western Blot

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Figure Lengend Snippet: P2Y 12 receptor in human cortex. Sections from human healthy and SPMS frontal cortex were analyzed by double immunofluorescence and confocal microscopy for the immunoreactive markers c-ter (red, panels (a), (c); insets a1, a2, c1; yellow merged, inset c2), intra1 (red, panels (d), insets d1, d2, d3; yellow merged, panel f, insets f1, f2), MBP (green, panels (b), (c), (e), insets c1, e1; yellow merged, panel (f), inset f1), MHC II (green, inset d3), and integrin α II/ β 3 (green, insets b2, e2; yellow merged, insets c2, f2). The asterisks show decreased P2Y 12 immunoreactivity in proximity to MS lesion.

Article Snippet: P2Y 12 receptor-ATTO-594 ( intra fl ) , Polyclonal , 125–142 , P2Y 12 receptor , 1 : 50 , Alomone.

Techniques: Immunofluorescence, Confocal Microscopy

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Figure Lengend Snippet: Regional distribution of P2Y 12 in proximity to MS lesions. Sections from SPMS frontal cortex were analyzed by double immunofluorescence and confocal microscopy for c-ter (red) and MHC II (green) immunoreactivity. In proximity to the demyelinating active cortical lesion expressing augmented positivity for MHC II, microglia gradually lose immunoreactivity for c-ter antibody. Microglia express differential immunoreactivity in the four chosen areas which are found inside (circled b-c) and around (circled a–d) a lesion.

Article Snippet: P2Y 12 receptor-ATTO-594 ( intra fl ) , Polyclonal , 125–142 , P2Y 12 receptor , 1 : 50 , Alomone.

Techniques: Immunofluorescence, Confocal Microscopy, Expressing

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Figure Lengend Snippet: Draw of microglial marker expression as a function of activation. Branched microglia are represented in blue and activated microglia in red. Iba1 , CD11b, and MHC II are mostly expressed in microglia throughout the different morphological states and their expression increases during activation (light blue to red color). P2Y 12 c-ter (light blue) and CD68 (red) are expressed, respectively, in branched or roundish/activated microglia.

Article Snippet: P2Y 12 receptor-ATTO-594 ( intra fl ) , Polyclonal , 125–142 , P2Y 12 receptor , 1 : 50 , Alomone.

Techniques: Marker, Expressing, Activation Assay

Journal: bioRxiv

Article Title: Central inhibition of P2Y 12 R differentially regulates survival and neuronal loss in MPTP-induced Parkinsonism in mice

doi: 10.1101/2021.02.03.256941

Figure Lengend Snippet: (A-B) Murine BV2 microglia cells were pretreated with PSB 0739 (500 nM, 30 min) (A) or with Y-27632 (10 μM, 30 min) (B) and were incubated with LPS (100 ng / mL, 60 min), ADP (10 μM, 5 min) or solvent (control) and phosphorylation of p38 MAPK at Threonine 180 / Tyrosine 182 were determined by immunoblotting. Graphs show the densitometric evaluation (n=3). (C) WT mice were treated with 20 mg / kg MPTP daily for five consecutive days, followed by treatment with 0.3 mg / kg PSB 0739, 50 mg / kg body weight per day fasudil or its vehicle. Shown are representative immuno-confocal microscopy images of brain slices isolated from WT mice stained with antibodies directed against CD68 (microsialin, red), phosphorylated-p38 MAPK (green), Iba1 (purple), DAPI (blue) and overlay image (merge). Scale bar: 100 μm, corresponds to 20 μm inset (D) Quantification of CD68 fluorescence intensity (left panel), phosphorylated-p38 MAPK fluorescence intensity (middle panel) and Iba1 fluorescence intensity (right panel) (n=5-7, left panel; n= 5-9, middle panel; n= 5-7, right panel). Data represent the mean ± SEM; *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001 (two-way ANOVA with Bonferroni’s post-hoc test (A, B, D)).

Article Snippet: Antibodies directed against phosphorylated p38 MAPK (Cat. No. 9211) and p38 MAPK (Cat. No. 9212) were purchased from Cell Signaling Technology, against tyrosine hydroxylase was from Sigma-Aldrich (Cat. No. AB152), against P2Y 12 -receptor was from AnaSpec (Cat. No. AS-55043A), against Iba1 was from Synaptic System (Cat. No. 234004), against CD68 was purchased from Bio-Rad Laboratories (Cat. No. MCA1957).

Techniques: Incubation, Solvent, Control, Phospho-proteomics, Western Blot, Confocal Microscopy, Isolation, Staining, Fluorescence

Journal: bioRxiv

Article Title: Central inhibition of P2Y 12 R differentially regulates survival and neuronal loss in MPTP-induced Parkinsonism in mice

doi: 10.1101/2021.02.03.256941

Figure Lengend Snippet: (A-B) WT mice were treated with 20 mg / kg MPTP daily for five consecutive days, followed by treatment with 0.3 mg / kg PSB 0739, 50 mg / kg body weight per day fasudil or its vehicle. Shown are representative immuno-confocal microscopy images of brain slices isolated from WT mice stained with antibodies directed against p38 MAPK (total; green), Iba1 (magenta), DAPI (blue) and overlay image (merge). Scale bar: 100 μm, corresponds to 20 μm inset (A). Quantification of p38 MAPK (total) fluorescence intensity (left panel) and Iba1 fluorescence intensity (right panel) (n=8-9, left panel; n= 8-12, right panel) (B) . Data represent the mean ± SEM; *, p ≤ 0.05; (two-way ANOVA, with Bonferroni’s post-hoc test (B)).

Article Snippet: Antibodies directed against phosphorylated p38 MAPK (Cat. No. 9211) and p38 MAPK (Cat. No. 9212) were purchased from Cell Signaling Technology, against tyrosine hydroxylase was from Sigma-Aldrich (Cat. No. AB152), against P2Y 12 -receptor was from AnaSpec (Cat. No. AS-55043A), against Iba1 was from Synaptic System (Cat. No. 234004), against CD68 was purchased from Bio-Rad Laboratories (Cat. No. MCA1957).

Techniques: Confocal Microscopy, Isolation, Staining, Fluorescence

Journal: bioRxiv

Article Title: Central inhibition of P2Y 12 R differentially regulates survival and neuronal loss in MPTP-induced Parkinsonism in mice

doi: 10.1101/2021.02.03.256941

Figure Lengend Snippet: Neurodegeneration during Parkinson’s disease causes the release of damage-associated molecular pattern molecules (DAMPs), such as ADP and ATP. The released nucleotides active P2Y 12 -receptor (P2Y 12 R) expressed on microglia, and subsequently through activation of the heterotrimeric G protein Gi results in the inhibitory regulation of adenylate cyclase. Decreased levels of cAMP leads to reduced protein kinase A (PKA) activity, which in turn results in reduced phosphorylation of RhoA at serine 188. Phosphorylation of RhoA at serine 188 negatively regulate RhoA activity; therefore, the decreased phosphorylation state results in increased RhoA and in turn, enhanced ROCK and p38 MAPK activity, which then leads to bolstered pro- and anti-inflammatory cytokine biosynthesis. Arrows indicate stimulation, blunt lines represent inhibition. Red arrows indicate decrease in concentration or activity, green arrows indicate increased enzymatic activity. PSB 0739 is a specific inhibitor of P2Y 12 R, Y-27632 is a specific inhibitor for ROCK.

Article Snippet: Antibodies directed against phosphorylated p38 MAPK (Cat. No. 9211) and p38 MAPK (Cat. No. 9212) were purchased from Cell Signaling Technology, against tyrosine hydroxylase was from Sigma-Aldrich (Cat. No. AB152), against P2Y 12 -receptor was from AnaSpec (Cat. No. AS-55043A), against Iba1 was from Synaptic System (Cat. No. 234004), against CD68 was purchased from Bio-Rad Laboratories (Cat. No. MCA1957).

Techniques: Activation Assay, Activity Assay, Phospho-proteomics, Inhibition, Concentration Assay