Journal: Scientific reports

Article Title: Primary mouse myoblast metabotropic purinoceptor profiles and calcium signalling differ with their muscle origin and are altered in mdx dystrophinopathy.

doi: 10.1038/s41598-023-36545-y

Figure Lengend Snippet: Figure 1. P2RYs transcript levels in myoblasts derived from Tibialis anterior (TA), Gastrocnemius (GC), Soleus (SOL) and Flexor Digitorum Brevis (FDB) muscles. Transcripts encoding P2RY2, P2RY4 and P2RY6 were tested in the same sample, thus their relative expression levels can be compared quantitatively., mRNAs encoding two ADP-activated receptors (P2RY1, P2RY12) were also detected in these samples. *p < 0.05, **p < 0.01 (mdx vs. w/t).

Article Snippet: The following primary antibodies were used: P2RY1 (1:270, APR-009), P2RY2 (1:270, APR-010), P2RY4 (1:300, APR-006), P2RY6 (1:250, APR-011), P2RY12 (1:270, APR-012), P2RY13 (1:270, APR017); all Alomone Labs, calsequestrin (ab126241), calreticulin (ab128885), SERCA1 (ab124501) and SERCA2 (ab91032); all Abcam, diluted 1:1000, Gαq11 (06-709 Merck Millipore, 1:1000,), PLCβ isoforms 3–4 (sc-133231, sc-166131, respectively; Santa Cruz Biotechnology, all diluted 1:100,), NCX1 (R3F1 Swant, 1:1000), NCX3 (ab84708 Abcam, 1:500), PMCA (ab2825 Abcam, 1:1000) and IP3R (#8568 Cell Signalling Technology, 1:1000).

Techniques: Derivative Assay, Muscles, Expressing

Journal: Scientific reports

Article Title: Primary mouse myoblast metabotropic purinoceptor profiles and calcium signalling differ with their muscle origin and are altered in mdx dystrophinopathy.

doi: 10.1038/s41598-023-36545-y

Figure Lengend Snippet: Figure 4. Western bloting analysis of ADP-activated metabotropic receptors P2RY12 and P2RY13. Each bar shows Western blot data from 3 independent experiments ± SD. Representative western blots are also shown. *p < 0.05 (mdx vs. w/t).

Article Snippet: The following primary antibodies were used: P2RY1 (1:270, APR-009), P2RY2 (1:270, APR-010), P2RY4 (1:300, APR-006), P2RY6 (1:250, APR-011), P2RY12 (1:270, APR-012), P2RY13 (1:270, APR017); all Alomone Labs, calsequestrin (ab126241), calreticulin (ab128885), SERCA1 (ab124501) and SERCA2 (ab91032); all Abcam, diluted 1:1000, Gαq11 (06-709 Merck Millipore, 1:1000,), PLCβ isoforms 3–4 (sc-133231, sc-166131, respectively; Santa Cruz Biotechnology, all diluted 1:100,), NCX1 (R3F1 Swant, 1:1000), NCX3 (ab84708 Abcam, 1:500), PMCA (ab2825 Abcam, 1:1000) and IP3R (#8568 Cell Signalling Technology, 1:1000).

Techniques: Western Blot

Journal: Purinergic Signalling

Article Title: Using antibodies against P2Y and P2X receptors in purinergic signaling research

doi: 10.1007/s11302-011-9278-z

Figure Lengend Snippet: Expression of P2Y receptors in Müller cells of the adult rat and mouse retinal slices. Immunohistochemical staining was accomplished using anti-P2Y 2 receptor (#APR-010) and anti-P2Y 6 receptor (#APR-011) antibodies. a No distinct immunoreactivity for the P2Y 2 receptor ( red ) was observed in Müller cells. However, ganglion cells ( asterisk ) and structures within the inner plexiform layer ( IPL ), inner nuclear layer ( INL ), and outer plexiform layer ( OPL ) display faint staining. b P2Y 4 receptor ( red ) is highly expressed in Müller cell end-feet ( arrows ), as detected using anti-P2Y 4 receptor antibody (#APR-006). Additionally, a band of globular structures, probably representing synapse of bipolar cells in the ON-sublamina ( ON ) of the IPL, is stained for this receptor. c The staining pattern of the P2Y 6 receptor ( red ) is similar to that of P2Y 2 . The insets in ( b ) and ( c ) show the ganglion cell layer ( GCL ) and the INL, respectively, at higher magnification. ONL outer nuclear layer, OFF OFF-sublamina of the IPL, PRS photoreceptor segments. Scale bars , 20 μm; inset scale bars , 10 μm. d Immunohistochemical staining was carried out using anti-P2Y 4 receptor antibody. The slices were co-stained against the glial cell marker cellular retinaldehyde-binding protein ( CRALBP ); co-labeling yielded a yellow-orange merge signal. Arrows , Müller cell end-feet; arrowheads , Müller cell somata. GCL ganglion cell layer, IPL inner plexiform layer, NFL nerve fiber layer, ONL outer nuclear layer, PRS , photoreceptor segments. Scale bars , 20 μm. a–c Adapted from reference 12 with permission of John Wiley and Sons. d Adapted from reference 13 with permission of Wiley-Blackwell

Article Snippet: In another study, the expression and function in nociception of P2Y 1 and P2Y 12 using their respective Alomone Labs antibodies via immunohistochemistry analysis was examined in sensory neurons [ ].

Techniques: Expressing, Immunohistochemical staining, Staining, Marker, Binding Assay, Labeling

Journal: Purinergic Signalling

Article Title: Using antibodies against P2Y and P2X receptors in purinergic signaling research

doi: 10.1007/s11302-011-9278-z

Figure Lengend Snippet: Differential expression of P2Y receptors in glial following retinal detachment. Immunohistochemical staining of control retinas and from detached areas of porcine retinas 7 days after surgery was done using anti-P2Y 1 receptor (#APR-009), anti-P2Y 2 receptor (#APR-010), and anti-P2Y 4 receptor (#APR-006) antibodies ( red ). Immunoreactivities for P2Y 1 , P2Y 2 , and P2Y 4 receptor proteins ( red ) coincide with that of intermediate filament constituents vimentin ( green and yellow for merging signal). Arrows , vimentin-positive fibers in the outer retina that were also stained for P2Y 2 protein. GCL ganglion cell layer, INL inner nuclear layer, IPL inner plexiform layer, NFL nerve fiber layer, ONL outer nuclear layer. Scale bars , 20 μm. Adapted from reference 15 with permission of Association for Research in Vision and Ophthalmology

Article Snippet: In another study, the expression and function in nociception of P2Y 1 and P2Y 12 using their respective Alomone Labs antibodies via immunohistochemistry analysis was examined in sensory neurons [ ].

Techniques: Expressing, Immunohistochemical staining, Staining

Journal: Purinergic Signalling

Article Title: Using antibodies against P2Y and P2X receptors in purinergic signaling research

doi: 10.1007/s11302-011-9278-z

Figure Lengend Snippet: Expression of P2Y receptors in rat distal colon. All P2Y immunohistochemical staining was done using Alomone Labs anti-P2Y receptor antibodies. a Double immunolabeling reveals that P2Y 1 is expressed in enteric smooth muscle cells and S100 in enteric glial cells. b In the myenteric plexus, P2Y 2 is detected in neurons. c P2Y 4 is detected in enteric glial cells. d P2Y 6 is expressed in vascular and enteric smooth muscle cells. e P2Y 11 is detected in enteric glial cells and P2Y 12 is expressed in enteric nerves ( f ). Adapted from reference 30 with permission of Elsevier

Article Snippet: In another study, the expression and function in nociception of P2Y 1 and P2Y 12 using their respective Alomone Labs antibodies via immunohistochemistry analysis was examined in sensory neurons [ ].

Techniques: Expressing, Immunohistochemical staining, Staining, Immunolabeling

Journal: Purinergic Signalling

Article Title: Using antibodies against P2Y and P2X receptors in purinergic signaling research

doi: 10.1007/s11302-011-9278-z

Figure Lengend Snippet: Expression of P2Y receptors in rat duodenal epithelium. Immunohistochemical staining was done using anti-P2Y 1 receptor antibody (#APR-009), anti-P2Y 2 receptor antibody (#APR-010), and anti-P2Y 6 receptor antibody (#APR-011). a P2Y 1 is expressed along the brush border membranes of villous cells. P2Y 2 and P2Y 4 receptors are not expressed ( b , c ). d P2Y 6 receptor is diffusely expressed in the villous cells and interstitium. Adapted from reference 31 with permission of John Wiley and Sons

Article Snippet: In another study, the expression and function in nociception of P2Y 1 and P2Y 12 using their respective Alomone Labs antibodies via immunohistochemistry analysis was examined in sensory neurons [ ].

Techniques: Expressing, Immunohistochemical staining, Staining

Journal: Purinergic Signalling

Article Title: Using antibodies against P2Y and P2X receptors in purinergic signaling research

doi: 10.1007/s11302-011-9278-z

Figure Lengend Snippet: Expression of P2Y receptors in osteoblasts. Using flow cytometry analysis, P2Y 1 , P2Y 2 , P2Y 4 , P2Y 6 , P2Y 11 , P2Y 12 , and P2Y 13 receptors were detected in osteoblasts using their respective Alomone Labs antibodies ( open histograms ). Incubating the antibody with the control peptide antigen was used as a negative control ( filled histograms ). Adapted from reference 41 with permission of Elsevier

Article Snippet: In another study, the expression and function in nociception of P2Y 1 and P2Y 12 using their respective Alomone Labs antibodies via immunohistochemistry analysis was examined in sensory neurons [ ].

Techniques: Expressing, Flow Cytometry, Negative Control

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