PPM1K Antibody - #DF4348

Figure 1. PP2Cm is downregulated in DRG neurons of HFD‐induced obese mice A. Growth curves of mice fed on chow diet (CD) or high fat diet (HFD). Mice were weighted weekly (n = 10 per group). B. Mechanical pain threshold indicated by von Frey filaments (n = 6 per group). C. The percentage of paw withdrawal responses to dynamic stimulation of brush (n = 6 per group). D. The BCAA levels in the L3‐L5 DRGs (n = 6 per group). E. Photograph of mice feeding on CD and HFD. F. Representative Western blot of PP2Cm in the L3‐L5 DRGs of mice after 10 weeks of diet intervention. G–J. Representative images of PP2Cm (green) co‐stained with neurons (NeuN, red) (G) or glia cells (FABP7, red) (H) in the L3‐L5 DGs; scale bar, 20 μm. The PP2Cm intensity are shown in the bar graphs (n > 50 cells per group) (I, J).

Figure 1. PP2Cm is downregulated in DRG neurons of HFD‐induced obese mice A. Growth curves of mice fed on chow diet (CD) or high fat diet (HFD). Mice were weighted weekly (n = 10 per group). B. Mechanical pain threshold indicated by von Frey filaments (n = 6 per group). C. The percentage of paw withdrawal responses to dynamic stimulation of brush (n = 6 per group). D. The BCAA levels in the L3‐L5 DRGs (n = 6 per group). E. Photograph of mice feeding on CD and HFD. F. Representative Western blot of PP2Cm in the L3‐L5 DRGs of mice after 10 weeks of diet intervention. G–J. Representative images of PP2Cm (green) co‐stained with neurons (NeuN, red) (G) or glia cells (FABP7, red) (H) in the L3‐L5 DGs; scale bar, 20 μm. The PP2Cm intensity are shown in the bar graphs (n > 50 cells per group) (I, J).

Fig. 2 Conditional knockout of PP2Cm in DRG neurons induces pain hypersensitivity a, Representative cartoons of pAAV9-hSyn-(Cre) injection. Intrathecal injection of pAAV9-hSyn-Cre or pAAV9-hSyn into male and female PP2Cmfl/fl mice resulted in the generation of PP2Cm-cKO and PP2Cm-ctrl mice. b, Representative immunoblots of PP2Cm in the L4-L5 DRGs on day 21 post-injection in PP2Cm-cKO and PP2Cm-ctrl mice of both sexes. male n = 3 experimental repeats (6 mice)/group, female n = 3 experimental repeats (6 mice)/group. c-e, Mechanical withdrawal thresholds (c), withdrawal latencies to heat (d) and cold stimulus (e) in PP2Cm-cKO and PP2Cm-ctrl mice. n = 10 mice/group, both sexes. f, BCAA concentrations in the L4-L5 DRGs of PP2Cm-cKO and PP2Cm-ctrl mice. n = 5 mice/group, both sexes. g-i, Mechanical withdrawal thresholds (g), withdrawal latencies to heat (h) and cold stimulus (i) in PP2Cm-cKO mice with or without BT2 supplementation after 7 consecutive days, PP2Cm-cKO + Vehicle vs. PP2Cm-cKO + BT2. n = 6 mice/group, both sexes. j, BCAA concentrations in the L4-L5 DRGs of PP2Cm-cKO + Vehicle and PP2Cm-cKO + BT2 mice. n = 6 mice/group, both sexes. Data are shown as the mean ± SEM. Statistical tests used were unpaired two-tailed Student’s t-test (c-e, g-i), one-way ANOVA with Tukey’s multiple comparisons test (b), and two-way repeated-measures ANOVA with Bonferroni’s post hoc test (f, j). *p 

Fig. 4 Inhibiting the CCL5/CCR5 signaling pathway attenuates hyperalgesia in DRG PP2Cm-deficient mice a, Quantification for release of CCL5 in the L4-L5 DRGs of PP2Cm-ctrl and PP2Cm-cKO mice, as detected by ELISA. n = 4 mice/group. b, Illustrative cartoon depicting the intrathecal administration of an anti-CCL5 neutralizing antibody. c, Quantification for release of CCL5 in the L4-L5 DRGs of PP2Cm-cKO + IgG, PP2Cm-cKO + anti-CCL5 1d and PP2Cm-cKO + anti-CCL5 3d mice. n = 4 mice/group. d-f, Mechanical withdrawal thresholds (d), withdrawal latencies to heat (e) and cold stimulus (f) on day 0 (baseline, BL), 1, and 3 post-injection in PP2Cm-cKO mice treated with IgG or anti-CCL5 neutralizing antibody. n = 5–6 mice/group. g, The L4-L5 DRGs from PP2Cm-ctrl and PP2Cm-cKO mice were double stained with PP2Cm (green) and CCR5 (red). The corner image in white square is the zoomed-in image of the area in the smaller white square. Scale bar: 20 μm. h, i, Fluorescent quantifications for PP2Cm (h) and CCR5 (i) in DRG sections from PP2Cm-ctrl and PP2Cm-cKO mice. n = 3 sections from 3 mice/group. j, A representative cartoon illustrating the intrathecal injection of CCR5 antagonist maraviroc or Ccr5 small interfering RNA (siCcr5). k-m, Mechanical withdrawal thresholds (k), withdrawal latencies to heat (l) and cold stimulus (m) at BL, 1 h, and 24 h post-injection in PP2Cm-cKO mice treated with Vehicle or maraviroc. n = 6 mice/group. n, The mRNA levels of Ccr5 in the L4-L5 DRGs from PP2Cm-cKO + siCtrl, PP2Cm-cKO + siCcr5 2d, and PP2Cm-cKO + siCcr5 8d mice (to β-actin). n = 3 experimental repeats (6 mice)/group. o-q, Mechanical withdrawal thresholds (o), withdrawal latencies to heat (p) and cold stimulus (q) at BL, 2d, and 8d post-injection in PP2Cm-cKO mice treated with siCtrl or siCcr5. n = 5–6 mice/group. Data are shown as the mean ± SEM. Statistical tests used were unpaired two-tailed Student’s t-test (a, h, i), one-way ANOVA with Tukey’s multiple comparisons test (c, n), and two-way repeated-measures ANOVA with Bonferroni’s post hoc test (d-f, k-m, o-q). *p 

Figure 2. HFD-induced hyperalgesia is coupled with an accumulation of BCAAs in DRG. (A) BCAA (leucine, isoleucine, and valine) levels in plasma after 4 and 8 weeks feeding in CD (n = 6) and HFD mice (n = 6). (B) BCAA levels in L4-L5 DRG under 4 and 8 weeks of CD (n = 6) and HFD diet (n = 6). (C,D) The linear regression analysis between mechanical (C) or thermal (D) withdrawal thresholds and BCAA levels (leucine + isoleucine + valine) in L4-L5 DRG under 8 weeks of diet. (E) Western blots analysis of BCAAs catabolic enzymes, PP2Cm, P-BCKDHS293 and BCKDH-E1α, in L4-L5 DRG after 8 weeks on CD (n = 3) or HFD (n = 3). (F) The BCKDH activity state in DRG after 8 weeks on CD (n = 3) or HFD (n = 3). Activity state means percentage of the BCKDH complex in the active dephosphorylated form. Data are shown as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.005 and ****P < 0.001.