Journal: JCI insight

Article Title: Age-related decline in hippocampal tyrosine phosphatase PTPRO is a mechanistic factor in chemotherapy-related cognitive impairment.

doi: 10.1172/jci.insight.166306

Figure Lengend Snippet: Figure 6. Amelioration of CRCI by hippocampal PTPRO is associated with inactivation of the SRC/EPHA4 axis. (A) Representative IHC images of PTPRO, p-SRC, SRC, p-EPHA4, EPHA4, p-EPHB2, and EPHB2 in the hippocampi. Scale bar: 50 μm. (B) The mean optical density of p-SRC/SRC, p-EPHA4/EPHA4, and p-EPHB2/EPHB2 in the hippocampi. (C) Immunoblotting of PTPRO, p-SRC, SRC, p-EPHA4, EPHA4, p-EPHB2, and EPHB2 in the hippocampi under CRCI. Data are representative of 3 independent experiments. (D) The mean optical density of p-SRC/SRC, p-EPHA4/EPHA4, and p-EPHB2/EPHB2 in the hippocampi. Error bars: SEM. NS, not significant; *P < 0.05, ***P < 0.001 by 2-sided Student’s t test.

Article Snippet: The membranes were immersed in blocking buffer (5% skim milk in PBS) for 1 hour at room temperature and incubated 1 9 R E S E A R C H A R T I C L E JCI Insight 2023;8(14):e166306 https://doi.org/10.1172/jci.insight.166306 overnight with primary antibodies used against the following proteins: PTPRO (1:1000; catalog 12161-1- AP, Proteintech Group), p-SRC (1:1000; catalog 2101, Cell Signaling Technology), SRC (1:1000; catalog sc-8056, Santa Cruz Biotechnology), p-EPHB2 (1:1000; catalog ab61791, Abcam), EPHB2 (1:1000; catalog 83029, Cell Signaling Technology), p-EPHA4 (1:1000; catalog EP2731, ECM Biosciences), EPHA4 (1:1000; catalog sc-365503, Santa Cruz Biotechnology), PSD95 (1:1000; catalog 3450, Cell Signaling Technology), Syp (1:1000; catalog ab32127, Abcam), β-actin (1:1000; catalog 4967, Cell Signaling Technology), and GAPDH (1:1000; catalog ab8245, Abcam).

Techniques: Western Blot

Journal: JCI insight

Article Title: Age-related decline in hippocampal tyrosine phosphatase PTPRO is a mechanistic factor in chemotherapy-related cognitive impairment.

doi: 10.1172/jci.insight.166306

Figure Lengend Snippet: Figure 7. Region-specific restoration of hippocampal PTPRO ameliorates DOX-induced CRCI in Ptpro–/– female mice. (A) Schematic of the experimental design. (B) Representative immunofluorescence image of FLAG (green) in the hippocampi of Ptpro–/– mice 2 weeks after injection of Lentivirus-hSyn-Pt- pro-3×Flag into the CA3 region. Scale bar: 200 μm. DG, dentate gyrus. (C) Changes in spontaneous alternation behavior in the Y-maze test. (D) Represen- tative swimming traces in the MWM test. (E and F) Training trials were performed in the MWM test, in which the time taken to reach the submerged plat- form (E) and the distances traveled before reaching the submerged platform (F) were assesssed, n = 10 per group. (G and H) A probe trial was performed in the MWM test. Shown are the time spent in the target quadrant (G) and the number of crossings before reaching the target location (H), n = 10 per group. (I) Immunoblotting of PTPRO, p-SRC, SRC, p-EPHA4, and EPHA4 in the mouse hippocampal CA3 region. Data are representative of 3 independent exper- iments. Error bars: SEM. NS, not significant; *P < 0.05, **P < 0.01, ***P < 0.001 by 2-way ANOVA followed by a Tukey-Kramer post hoc test (C, G, and H). *Ptpro+/+-LVCon vs. Ptpro–/–-LVCon; #Ptpro–/–-LVCon vs. Ptpro–/–-LVPtpro; *P < 0.05, ***P < 0.001; #P < 0.05 by 3-way ANOVA followed by a Tukey-Kramer post hoc test (E and F). All values and statistical analysis of behavioral experiments are provided in Supplemental Table 4.

Article Snippet: The membranes were immersed in blocking buffer (5% skim milk in PBS) for 1 hour at room temperature and incubated 1 9 R E S E A R C H A R T I C L E JCI Insight 2023;8(14):e166306 https://doi.org/10.1172/jci.insight.166306 overnight with primary antibodies used against the following proteins: PTPRO (1:1000; catalog 12161-1- AP, Proteintech Group), p-SRC (1:1000; catalog 2101, Cell Signaling Technology), SRC (1:1000; catalog sc-8056, Santa Cruz Biotechnology), p-EPHB2 (1:1000; catalog ab61791, Abcam), EPHB2 (1:1000; catalog 83029, Cell Signaling Technology), p-EPHA4 (1:1000; catalog EP2731, ECM Biosciences), EPHA4 (1:1000; catalog sc-365503, Santa Cruz Biotechnology), PSD95 (1:1000; catalog 3450, Cell Signaling Technology), Syp (1:1000; catalog ab32127, Abcam), β-actin (1:1000; catalog 4967, Cell Signaling Technology), and GAPDH (1:1000; catalog ab8245, Abcam).

Techniques: Immunofluorescence, Injection, Western Blot