Fig 1: Knockdown of Dbi induces mechanical hypersensitivity in mice.A–E, siRNA against Dbi (or a non-targeting control siRNA) were intrathecally injected (2 μg/site) and 48 h later the following tests were performed: mechanical sensitivity (von Frey) test (A), cold allodynia (dry ice) test (B), Hargreaves test (C), Brush test (D), Alligator clip test (E). Bars are mean ± S.E.M.; ** indicates significant difference with p<0.001 for groups indicated by the connector line (unpaired t-test). F, Recovery of mechanical hypersensitivity (von Frey test) induced by the intrathecal siRNA knockdown of DBI with the intrathecal injection of recombinant DBI (10 ng/site). ### indicate significant difference from baseline (p<0.001); **, *** indicate significant difference from time-matched saline group (p<0.01, p<0.001; two-way repeated-measures ANOVA with Sidák post-hoc test). G, Schematic timeline for the viral DRG gene delivery and osmotic mini-pump experiment; inset depicts DRG 8 weeks after injection with AAV9-U6-shDBI-CAG-EGFP virions. H, Mechanical sensitivity was monitored with the von Frey test during 42 days after the DRG injection of AAV9-U6-shDBI-CAG-EGFP virions or GFP control virions (1.1 – 1.2×1012 vg/ml; 2 μl). *** indicate significant difference from time-matched control group (p<0.001; two-way repeated-measures ANOVA with Tukey’s post-hoc test). I, Similar to H but heat sensitivity was tested with the Hargreaves test. J, K, Mechanical (J) and heat (K) sensitivity was monitored after the implantation of osmotic mini-pumps delivering recombinant DBI to the DRG (200 μM, 0.5 μl/h; see Methods) to the mice pre-injected with the AAV9-U6-shDBI-CAG-EGFP virions.
Fig 2: DBI is a positive allosteric modulator (PAM) at presumed-mechanosensitive DRG neurons.Shown are the results of the perforated patch recordings from cultured rat DRG neurons. A, B, Determinants of a putative mechanosensitive DRG neuron: narrow action potential with a single minimum in the first derivative (A) and no repose to 1 μM capsaicin. An example of concentration-dependency of responses to GABA (1 – 5000 μM) is shown in B. C, D, Absolute (C) and normalised (D) concentration dependency of GABA currents (quantified as current densities) in the absence (black) and presence (blue) of 500 nM DBI. *indicates significant difference from concentration-matched control group (p<0.01; two-way repeated-measures ANOVA with Bonferroni post-hoc test). E, F, Summaries for the EC50 (E) and Hill coefficient (F) for experiments exemplified in B–D. * indicates significant difference for the Hill Coefficient between the indicated groups (p<0.05; unpaired t-test). G, H, Determinants of a putative polymodal nociceptor: broad action potential with two minima in the first derivative (G) and robust response to 1 μM capsaicin. An example of concentration-dependency of responses to GABA (1–5,000 μM) is shown in H. I–L, Similar to panels C-F but for the putative nociceptors.
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