P2Y14 Antibody PA5-34087 from Thermo Fisher Scientific

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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: 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 4: 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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eLife
March 21 2022

Fig 5: 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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Thermo Fisher Scientific for
P2Y14 Antibody

The P2Y14 Antibody from Thermo Fisher Scientific is a Rabbit Polyclonal antibody to P2RY14. This antibody recognizes Homo Sapiens, and Human antigen. The P2Y14 Antibody has been validated for the following applications: Immunocytochemistry, Immunohistochemistry, and Immunohistochemistry - fixed.

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P2Y14 Antibody from Thermo Fisher Scientific

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P2Y14 Antibody from Thermo Fisher Scientific

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