Fig 2: Phospho-BTK is localized to microglia 3 days following SCI, measured by immunofluorescence imaging. Photomicrographs of representative transverse spinal sections taken from spinal cord injured rat. Double immunofluorescent imaging analysis of spinal cord sections at lesion site showed phospho-BTK (red) expressed in microglia (Iba1, green) 3 days post-injury. The sections were immunostained with a primary antibody against phospho-BTK (red), and Iba1 (green) and counterstained with DAPI to identify cell nuclei. Phospho-BTK immunostaining was uniformly observed in cells immunoreactive for Iba1 (a specific calcium-binding protein for activation of microglia and macrophages [54]). This is from 3 days post SCI, at which time the Iba1 cells are predominantly microglia. The SCI conditions are as described in other figures. The images were obtained from the ventral white matter, in a section rostral 1 mm from the lesion epicenter using the Nikon confocal microscopy system (Nikon C2+, Melville, NY, USA) in the Spinal Cord and Brain Injury Research Center imaging core at the University of Kentucky. Scale bar: 10 µm.

Fig 3: Effects of treadmill exercise on activated microglia in hippocampi of APP/PS1 mice. (A) Flow cytometry analysis of microglia. Pacific Blue-A and FITC-A represent CD11b and CD45, respectively. P2 represents CD11b+CD45+cells, which are activated microglia. (B) Quantitative analysis of CD11b+CD45+cells in three groups. (C) Immunofluorescence (IF) of microglia and Aß in WC, TC and TE group, the Iba1 staining was performed to mark the microglia. (D) Statistic analysis of the number of microglia and Aß protein double positive cells in each group. n = 6 per group. **p < 0.01 vs. WC; &p < 0.05 vs. TC.

Fig 4: Effects of Ibrutinib post-treatment on Iba1 and GFAP proteins analyzed by quantification of Western blotting data. Western blot analysis of spinal cord samples (60 µg of protein extract each sample) at lesion epicenter showed that contusion injury increased Iba1 and GFAP activity in the spinal cord seven days post-injury compared with sham-operated animals. Ibrutinib treatment (6 mg/kg/day, starting at 3 h post-injury for seven days) resulted in reduced levels of Iba1 and GFAP in the spinal cord lesion site at 7 days after contusive SCI compared with vehicle-treated animals. Quantification of Iba1/GADPH and GFAP/GADPH seven days after contusive SCI was performed by the fold of blot density. Antibody was specific for the Iba1 or GFAP. Data are presented as mean ± S.E.M., n = 4 per group, and analyzed with one-way ANOVA followed by Bonferroni post hoc analysis, * p < 0.05, ** p < 0.01 compared to vehicle treated SCI animals.

Fig 5: CETN2 specifically labels astrocytes. (A–A”) CETN2 labeling overlaps with canonical astrocytic marker GFAP in human prefrontal cortex sections. (B–D) CETN2 specificity is confirmed by a lack of co-localization with neuronal protein RNA-binding fox-1 homolog 3 (RBFOX3), microglial marker ionized calcium-binding adaptor molecule 1 (IBA1), and mature oligodendrocyte marker adenomatous polyposis coli (APC). (E–H) Scatterplot correlation graphs showing robust co-localization of CETN2 with GFAP and poor overlap with RBFOX3, as well as with IBA1 and APC. Pearson’s coefficients are indicated on each plot. (I) Histogram shows CETN2 co-localization percentages with other neuronal and non-neuronal cell types. Data represent mean ± SEM. Scale bar: (A–D) 20 µm.