Anti-P2Y14 Receptor (extracellular) Antibody APR-018 from Alomone Labs, Ltd.

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eLife
March 21 2022

Fig 1: Mouse Nf1 mutant Schwann cell precursors (SCPs) use P2RY14 signaling to regulate self-renewal.(A) Quantification of percent of sphere forming cells in mouse wild-type (WT) and Nf1-/- SCPs treated with the selective P2ry14 inhibitor (300 nM 4-[4-(4-piperidinyl)phenyl]-7-[4-(trifluoromethyl)phenyl]-2-naphthalenecarboxylic acid [PPTN]) (primary, secondary, tertiary passage) (n = 3; two-way ANOVA; primary: *p = 0.0375, ***p = 0.0001, ****p < 0.0001; secondary: *p = 0.0101, **p = 0.0015, ***p = 0.0009; tertiary: *p = 0.0101, **p = 0.0050, ***p = 0.0005). (B) P2ry14 mRNA expression in WT and Nf1-/- E12.5 mouse SCP treated with sh non-target (shNT) control and shP2ry14 (****p < 0.0001). (C) Western blot of WT and Nf1-/- SCPs treated with shNT and shP2ry14 showing P2ry14 knockdown. WT shP2ry14 show a 0.4-fold decrease of P2ry14 protein compared to WT shNT. Nf1-/- shP2ry14 show a 0.5-fold decrease compared to Nf1-/-. (D) Quantification of percent of sphere forming cells in mouse WT and Nf1-/- SCPs treated with shNT and shP2ry14 (n = 3; two-way ANOVA; primary: *p = 0.0288, ****p < 0.0001; secondary: **p = 0.0029,***p = 0.0005, ****p < 0.0001; tertiary: *p = 0.0154, ***p = 0.0007). (E) Western blot of WT and Nf1-/- Schwann cell (SC) spheres shows changes in pPKA substrate phosphorylation after shP2ry14 knockdown. WT shP2ry14 shows a 1.43-fold increase in pPKA after P2ry14 knockdown. Nf1-/- cells have a 1.31-fold increase in pPKA expression after P2ry14 knockdown. (F) Quantification of percent of sphere forming cells in Nf1-/- mouse SCPs treated with 1 µM rolipram or 300 nM PPTN (n = 3; two-way ANOVA; primary: ***p = 0.0002, ****p < 0.0001; secondary **p = 0.0030, ****p < 0.0001; tertiary: *p = 0.0476, ***p = 0.0004).
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eLife
March 21 2022

Fig 2: P2RY14 deletion improves nerve ultrastructure.(A) Electron micrograph of saphenous nerve of 4-month-old wild-type (WT), Nf1fl/fl;DhhCre, P2ry14-/-; Nf1fl/fl;DhhCre and P2ry14-/- mice. (B) Electron micrograph of saphenous nerve of 7-month-old WT, Nf1fl/fl;DhhCre and P2ry14-/-;Nf1fl/fl;DhhCre mice. (C) Remak bundle quantification at 4 months of age (n = 3; two-way ANOVA: ****p < 0.0001). (D) Remak bundle quantification at 7 months of age (n = 3; two-way ANOVA: **p = 0.0027, ****p < 0.0001).
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eLife
March 21 2022

Fig 3: P2RY14 is expressed in Schwann cell precursors (SCPs) and Schwann cells (SCs) in vivo in a mouse model of neurofibromatosis.(A) Schematic of generation of neurofibroma mice breeding of P2ry14-/- mice with Nffl/fl mice to obtain P2ry14+/-; Nf1fl/fl;DhhCre and P2ry14-/-; Nf1fl/fl;DhhCre littermates after several crosses. (B) Genotyping confirmation of wild-type (WT) and P2ry14 knockout (KO) alleles. (C) Western blot of sciatic nerve and neurofibroma tumors of Nf1fl/fl;DhhCre and P2ry14-/-;Nf1fl/fl;DhhCre mice show decrease in P2ry14 expression upon P2ry14 knockdown (1- to 0.1-fold decrease in sciatic nerve and 1- to 0.2-fold decrease in neurofibroma tissue). (D) Spinal cord (SC) immunofluorescent staining of mouse embryos at embryonic day 12.5 (E12.5) shows P2ry14 expression (ß-galactosidase) co-localization with SOX-10 SCs at the dorsal root ganglion (DRG) and ventral roots (VR). (E) Spinal cord (SC) immunofluorescent staining of mouse embryos at E12.5 shows P2ry14 expression (ß-galactosidase) co-localization with SOX-10 SCs at DRG. E1 and E2 insets show enlarged sections of the DRG. (F) Immunofluorescent staining of 7-month-old mouse sciatic nerve shows ß-galactosidase positive staining as a confirmation of P2ry14 knock-in; co-labeling of ß-galactosidase and CNPase shows that P2ry14 co-localizes with SCs (inset). (G) DAB staining of 7-month-old WT nerve and Nf1fl/fl;DhhCre mouse neurofibromas (DAB staining: brown [P2ry14 positive cells] blue [cell nuclei]).
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eLife
March 21 2022

Fig 4: Cyclic AMP (cAMP) increase in a mouse model of neurofibromatosis either by rolipram or P2RY14 inhibitor treatment decreases Schwann cell (SC) proliferation.(A) Rolipram drug treatment experimental design. (B) Tumor lysates of vehicle and rolipram treated Nf1fl/fl;DhhCre mice show changes in p-PKA substrate. (C) Ki67 staining at 9 months of age in vehicle and rolipram treated mice. (D) Quantification of Ki67+ cells in vehicle treated versus rolipram treated mice (unpaired t-test: ****p < 0.0001; n = 3). (E) P2ry14 inhibitor (4-[4-(4-piperidinyl)phenyl]-7-[4-(trifluoromethyl)phenyl]-2-naphthalenecarboxylic acid [PPTN]) drug treatment experimental design. (F) Immunofluorescent staining of sciatic nerve of 4-month-old wild-type (WT), Nf1fl/fl;DhhCre (vehicle) and Nf1fl/fl;DhhCre (PPTN treated) shows increased p-PKA expression after PPTN treatment. (G) Ki67+ staining of Nf1fl/fl;DhhCre (vehicle) and Nf1fl/fl;DhhCre (PPTN treated) neurofibroma tissue. (H) Quantification of Ki67+ cells after PPTN treatment in neurofibroma tissue.
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eLife
March 21 2022

Fig 5: P2RY14 deletion in a mouse model of neurofibroma increases survival and delays neurofibroma initiation.(A) Kaplan-Meier survival plot of Nf1fl/fl;DhhCre (red line; n = 8); P2ry14+/-; Nf1fl/fl;DhhCre (black line, n = 14); P2ry14-/-; Nf1fl/fl;DhhCre (blue line; n = 13); Nf1fl/+ control (green line, n = 11) (*p = 0.0256) shows P2ry14-/-;Nf1fl/fl;DhhCre have increased survival. (B) Representative image of gross dissection of Nf1fl/fl;DhhCre and P2ry14-/-;Nf1fl/fl;DhhCre mice at 7 months of age. (C) Neurofibroma tumor number quantification at 7 months of age (unpaired t-test ****p < 0.0001). (D) Neurofibroma diameter quantification at 7 months (unpaired t-test ***p = 0.0004) (for C and D: Nf1fl/fl;Dhh+ n = 8 mice, 48 neurofibroma tumors; P2ry14-/-;Nf1fl/fl;Dhh+ n = 8 mice, 11 neurofibroma tumors). (E) Ki67 staining of mouse dorsal root ganglion (DRG) and neurofibroma tumor tissue at 7 months of age. (F) Quantification of Ki67+ cells in mouse DRG and neurofibroma tumor tissue at 7 months of age (one-way ANOVA; multiple comparisons ***p = 0.0008; ****p < 0.0001). (G) Anti-p-PKA substrate staining in wild-type (WT), Nf1fl/fl;DhhCre and P2ry14-/-; Nf1fl/fl;DhhCre mice. p-PKA substrate phosphorylation labeling co-localized with CNPase Schwann cell (SC) marker (insets).

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Anti-P2Y14 Receptor (extracellular) Antibody

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Alomone Labs, Ltd.'s Anti-P2Y14 Receptor (extracellular) Antibody is a Rabbit Polyclonal antibody. This antibody has been shown to work in applications such as: Live Cell Imaging, Immunofluorescence, Immunohistochemistry, and Western Blot. The Anti-P2Y14 Receptor (extracellular) Antibody was generated using P2RY14 as the antigen and it reacts with Human, Mouse, and Rat.

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Anti-P2Y14 (extracellular) Antibody (#APR-018) is a highly specific antibody directed against an epitope of the human protein. The antibody can be used in western blot, immunohistochemistry, and indirect flow cytometry applications. The antibody recognizes an extracellular epitope and is thus highly suited for detecting the receptor in living cells. It has been designed to recognize P2Y14 from human, rat, and mouse samples

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Anti-P2Y14 Receptor (extracellular) Antibody from Alomone Labs, Ltd.

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Anti-P2Y14 Receptor (extracellular) Antibody from Alomone Labs, Ltd.

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