Fig 2: P2X4R scFv reverses mechanical hypersensitivity in male mice. (A). A single dose of two P2X4R scFvs (95 or 12) given 3 weeks after induction of FRICT-ION model permanently reverses mechanical hypersensitivity in male mice through to the end of the seven week study time course (n = 6, two-way ANOVA (Dunnett’s multiple comparisons test)). (B). The scFv had no effect on responses of female mice with FRICT-ION-induced mechanical hypersensitivity (n = 4). (C). Blocking peptide targeting the same P2X4R peptide fragment significantly reduced hypersensitivity, increasing the mechanical threshold 4 hours after treatment but the effect was short-lived. * p < 0.05, *** p < 0.001, and **** p < 0.0001.

Fig 3: mAb27 staining of human cell lines shows a similar pattern as commercial (polyclonal) anti-hP2X4 Abs. The human prostate cancer cell line DU145 (A) or immortal human bronchial epithelial cells (16HBE14o–) (B), were stained with mAb 27 or with two polyclonal anti-P2X4 Abs from Santa Cruz and Abnova. P2X4 localization was visualized by confocal microscopy. Staining with secondary antibodies alone was negative (Figure S2). The images are representative of three independent experiments.

Fig 4: Isolation and binding efficacy of P2X4R scFv antibodies. (A). Analysis of RT-PCR recovery of VH/K cDNA from P2X4R peptide-immunized spleen in the 1st, 2nd, and 3rd cycles. (B). The diagram to the left illustrates the size difference between scFv and full IgG molecules. On the right are Western blots of three purified unique P2X4R scFvs generated by the cell-free ribosome display platform. (C). Plot of ELISA data showing nanomolar binding affinity for the three scFv antibodies with the best binding. (D). Binding specificity and cross-reactivity of three P2X4R scFvs to P2X4R, but not to neutral IgGs or scFvs as negative controls.

Fig 5: ATP released from activated Müller cells induces microglia activation through P2X7R. a–c Representative immunoblots showing the changes of GFAP and TSPO expression in retinas with intravitreal injections of DHPG (100 µM, 2 µL) (a), DHPG along with Gap26 (200 µM, 2 µL) (b), and DHPG along with BBG (10 µM, 2 µL) (c) at different times after the injections. d, e A comparison of average relative densitometric quantifications of the immunoreactive bands obtained following different kinds of injection is shown in the bar charts (d for GFAP; e for TSPO). f Representative immunoblots showing the changes of GFAP and TSPO expression at different post-operational times in normal and COH retinas with intravitreal injections of saline (2 µL)/20 µM MPEP (2 µL). Saline or MPEP injections were made two days before COH operation. g, h A comparison of average relative densitometric quantifications of the immunoreactive bands obtained following different kinds of injection is shown in the bar charts (g for TSPO; h for GFAP). i Representative immunoblots showing the changes in TSPO and GFAP protein expression in COH retinas with or without the intravitreal injection of BBG. 2 µL BBG (10 µM) or normal saline was intravitreally injected 2 day before the COH operation. j, k A comparison of average relative densitometric quantifications of the immunoreactive bands obtained following different kinds of injection is shown in the bar charts (j for TSPO; k for GFAP). l, m Western blot results showing the changes in TSPO protein expression in COH retinas with or without the intravitreal injection of 5-BDBD, a P2X4R antagonist. All the data are normalized to control. In all experiments, n = 6 for each group. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. Ctr using ordinary one-way ANOVA with Dunnett’s multiple comparisons test (d, e, g, h, j, k, m); #P < 0.05 and ###P < 0.001 vs. corresponding saline group at the same time point using paired two-tailed Student’s t test (g, h, j, k)