Changes in cellular specificity of endogenous PTEN in the dorsal horn of the spinal cord. (A) Western blots for phospho-PTEN, PTEN, and β-actin proteins from sham-operated and 14 days after CCI groups; (B) relative density of immunoblot of phospho-PTEN; (C) relative density of immunoblot of PTEN. Relative band intensities of phospho-PTEN and PTEN were quantified by densitometry and indicated as the percent change relative to that for the sham-operated group (100%). Western blotting of spinal dorsal horn tissue homogenate revealed that phospho-PTEN and PTEN protein expression was detectable in the sham-operated group, but CCI evoked upregulation of phospho-PTEN protein and downregulation of PTEN protein 14 days after CCI. Western blotting of β-actin was performed to verify the equivalent amounts of protein were loaded in each lane. Confocal double-immunofluorescent staining of PTEN (red) with NeuN (neuronal-specific marker; (D), green), GFAP (astrocyte specific marker; (E), green) OX-42 (microglial specific marker; (F), green) in the dorsal horn region of the lumbar spinal cord of sham-operated group. The merged images of (D), (E), and (F) (yellow; white arrow) indicate co-localization of PTEN with NeuN, GFAP, and OX-42 immunoreactive cells in the spinal cord, respectively. The confocal results showed that spinal PTEN was primarily co-localized with astrocytes. Scale bars are 50 μm for all images (D-F). Each bar in (B) and (C) represents the mean ± SEM with three rats per group. CCI-14 day: 14 days after CCI. *P < 0.05 compared with sham-operated group. CCI, chronic constriction injury; PTEN, phosphatase and tensin homolog deleted from chromosome 10; p-PTEN, phosphorylated PTEN.