- 702516 - Invitrogen Antibodies P2RY12 antibody

Submitted references Crosstalk between astrocytes and microglia results in increased degradation of α-synuclein and amyloid-β aggregates.

Rostami J, Mothes T, Kolahdouzan M, Eriksson O, Moslem M, Bergström J, Ingelsson M, O'Callaghan P, Healy LM, Falk A, Erlandsson A
Journal of neuroinflammation 2021 Jun 3;18(1):124

Inhibiting P2Y12 in Macrophages Induces Endoplasmic Reticulum Stress and Promotes an Anti-Tumoral Phenotype.

Pavlović N, Kopsida M, Gerwins P, Heindryckx F
International journal of molecular sciences 2020 Oct 31;21(21)

Elevated protein synthesis in microglia causes autism-like synaptic and behavioral aberrations.

Xu ZX, Kim GH, Tan JW, Riso AE, Sun Y, Xu EY, Liao GY, Xu H, Lee SH, Do NY, Lee CH, Clipperton-Allen AE, Kwon S, Page DT, Lee KJ, Xu B
Nature communications 2020 Apr 14;11(1):1797

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of P2Y12 was achieved by transfecting PC3 cells with P2Y12 specific siRNA (Silencer® select Product # s34983 and Product # s34985). Western blot analysis was performed using TEB extract from the P2Y12 knock down cells (lane 3), non-specific scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blots were probed with Anti-P2Y12 Recombinant Rabbit Monoclonal Antibody (Product # 702516, 1-2 µg/mL) and Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.4 µg/mL, 1:2500 dilution). Reduction of signal upon siRNA mediated knock down confirms that antibody is specific to P2Y12.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis was performed on Tissue extracts (30 µg lysate) of Rat Brain (Lane 1), Mouse Brain (Lane 2) and membrane extracts of U-87 MG (Lane 3), MCF-7 (Lane 4), MDA-MB-231 (Lane 5), PC-12 (Lane 6), IMR32 (Lane 7), SK-N-AS (Lane 8) and C8-D1A (Lane 9). The blots were probed with Anti-P2Y12 Recombinant Rabbit Monoclonal Antibody (Product # 702516, 1 µg/mL). A 60 kDa band corresponding to P2Y12 was observed across the cell lines and Tissues tested. The blots were detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.4 µg/mL, 1:5000 dilution). Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 4-12% Bis-Tris gel (Product # NP0321BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane with iBlot® Dry Blotting System (Product # IB21001). The membrane was probed with the relevant primary and secondary Antibody following blocking with 5% skimmed milk. Chemiluminescent detection was performed using Pierce™ ECL Western blotting Substrate (Product # 32106).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescent analysis of P2Y12 (red) in human iPSC-derived microglia. The cells were fixed with 4% paraformaldehyde for 15 min at room temperature, and then permeabilized and blocked with 0.25% Triton X-100 and 10% donkey serum in PBS for 20 min. Samples were then incubated with Anti-P2Y12 Recombinant Rabbit Monoclonal Antibody (red; Product # 702516) at a dilution of 1:500 in PBS containing 0.25% Triton X-100 and 10% donkey serum at 48304C overnight, followed by incubation with Donkey Anti-Rabbit Alexa Fluor 568 (Product # A10042) at a dilution of 1:1000 as well as DAPI (blue; 1:25000) in PBS containing 0.25% Triton X-100 and 10% donkey serum at room temperature for 1 hour. Images were taken at 20X magnification. (Scale bar: 50 µm) Data courtesy of Dr. Zhexing Wen at Emory University.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 1 The P2Y12 receptor is expressed in mouse models for liver cirrhosis and hepatocellular carcinoma. ( A ) Immunohistochemical staining with P2Y12 antibodies of liver sections from healthy mice, mice with carbon tetrachloride (CCL 4) -induced liver cirrhosis and mice with diethylnitrosamine (DEN)-induced liver cancer. ( B ) Quantification of immunohistochemical staining with P2Y12 antibodies. ( C ) Co-staining of P2Y12 and macrophage marker F4/80 in liver slides from DEN-induced hepatocellular carcinoma (HCC). ( D ) Immunocytochemical staining of THP1-differentiated macrophages and Raw264.7 cells. ( E ) Western Blot image of P2Y12 protein expression and beta-actin protein in THP1-differentiated macrophages and Raw264.7 cells. ( F ) mRNA expression levels of P2yr12 in interferon gamma and lipopolysaccharide-stimulated anti-tumoral (M1) macrophages and interleukin (IL)-13 and IL-4-stimulated pro-tumoral (M2) macrophages ( G ) Quantification of protein expression in pro-tumoral (M2) and anti-tumoral (M1) macrophages. ( H ) Representative image of Western blot showing P2Y12 protein expression in pro-tumoral (M2) and anti-tumoral (M1) macrophages. ( I ) mRNA expression of P2yr12 in livers from healthy mice, mice with CCL4-induced liver cirrhosis and mice with DEN-induced liver cancer. * = p < 0.05; ** = p < 0.01.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 2 Expression of P2Y12 is located in the stroma of patients with hepatocellular carcinoma. ( A ) Micrographs of hepatocellular carcinoma biopsies stained with P2Y12 antibodies, obtained from the Human Protein Atlas, marking tumor (T) and stomal (S) area [,]. ( B ) Detail of stroma and ( C ) tumor areas within the biopsies []. ( D ) Kaplan-Meier survival curve of HCC patients with high or low expression of P2Y12 obtained through the Human Protein Atlas []. ( E ) P2Y12 expression in different HCC stages, measured as number fragments per kilobase of exon per million reads (RPKM) from the Human Protein Atlas [].

702516

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