pmecp2 s421  (Novus Biologicals)

Journal: Aging and Disease

Article Title: Prelimbic Cortical Stimulation with L-methionine Enhances Cognition through Hippocampal DNA Methylation and Neuroplasticity Mechanisms

doi: 10.14336/AD.2022.0706

Figure Lengend Snippet: Protracted MET treatment and PrL DBS enhances neuroplasticity and hippocampal neurogenesis through the PKA-CaMKIIα-BDNF pathway. Representative Western blot images of neuroplasticity-associated proteins (A). Densitometric measurement by two-way ANOVA analysis of PSD95 (Treatment, p = 0.038; Stimulation, p = 0.01; Treatment x Stimulation, p = 0.01), SYP (p > 0.376), CREB (p > 0.369), pCREB (Treatment, p = 0.044) and pCREB/CREB ratio (Treatment, p = 0.074) showed enhanced neuroplasticity in MET and DBS animals (B). Representative Western blot images of proteins involved in memory neuroepigenetics (C). Two-way ANOVA revealed a significant stimulation effect (p = 0.041) for DNMT3a, insignificant treatment (p = 0.071) and significant stimulation (p = 0.041) effects for CaN, and significant treatment (p = 0.022) and insignificant stimulation (p = 0.068) effects for BDNF. No difference in the pMeCP2/MeCP2 ratio was observed among all groups (p > 0.205) (D). Representative Western blot images of proteins involved in the intermediate pathways of CREB phosphorylation (E). Two-way ANOVA showed no changes in pERK/ERK ratio (p > 0.499), significant changes in pPKA/PKA ratio (treatment, p = 0.048; stimulation, p = 0.046) and pCaMKIIα/CaMKIIα ratio (Treatment, p = 0.006; Stimulation, p = 0.004). (F). Scatter plot displaying significant correlations between CaN and pCaMKIIα/CaMKIIα ratio in SAL-DBS and MET-DBS animals, indicating that the inhibition of CaN by PrL DBS was strongly associated with the activation of CaMKIIα in the hippocampus. All groups: n=9. (G). Neurogenesis quantification by flow cytometry analysis for BrdU. Scatter plot displaying a selection of nuclei stained with DAPI, subsequent gating for BrdU, and removal of events doubly stained for GFAP and NeuN. Quantification of all groups revealed a significant main effect of stimulation (p = 0.012). More BrdU-positive events were found in SAL-DBS and MET-DBS animals compared to SAL-SHAM animals (H). Scatter plot displaying significant positive correlations between BrdU-positive cell count and BDNF protein expression in MET-DBS animals. SAL-SHAM: n=6; SAL-DBS: n=10; MET-SHAM: n=7; MET-DBS: n=10. (I). *, p<0.05; **, p<0.01; n.s., not significant. Data presented as mean ± s.e.m.

Article Snippet: Antibodies against PSD95 (1:1000, Cat# ab18258, Abcam), synaptophysin (1:1000, Cat# 5461, Cell Signaling Technology), CREB (1:1000, Cat# 9197, Cell Signaling Technology), pCREB (1:1000, Cat# 9198, Cell Signaling Technology), BDNF (1:1000, Cat# NB100-98682, Novus Biologicals), PKA C-α (1:1000, Cat# 4782, Cell Signaling Technology), pPKA C Thr197 (1:1000, Cat# 4781, Cell Signaling Technology), ERK1/2 (1:1000, Cat# 9102, Cell Signaling Technology), pERK1/2 T202/Y204 (1:1000, Cat# 9101, Cell Signaling Technology), CaMKIIα (1:1000, Cat# 3362, Cell Signaling Technology), pCaMKIIα Thr286 (1:1000, Cat# 12716, Cell Signaling Technology), MeCP2 (1:1000, Cat# NB600-1101, Novus Biologicals), pMeCP2 S421 (1:1000, Cat# NBP2-29524, Novus Biologicals), DNMT3a (1:1000, Cat# 3598, Cell Signaling Technology), Calcineurin A (1:1000, Cat# 2614, Cell Signaling Technology) and GAPDH (1:1000, Cat# 2118, Cell Signaling Technology) were used.

Techniques: Western Blot, Phospho-proteomics, Inhibition, Activation Assay, Flow Cytometry, Selection, Staining, Cell Counting, Expressing

Journal: Cancer Cell International

Article Title: Epigenetic regulation of osteopontin splicing isoform c defines its role as a microenvironmental factor to promote the survival of colon cancer cells from 5-FU treatment

doi: 10.1186/s12935-020-01541-z

Figure Lengend Snippet: Evaluation of OPNc expression associated with MeCP2 protein phosphorylation in clinical colorectal cancer tissues. a Relative mRNA levels of OPN splicing isoforms in colon cancer tissues (n = 325) and the adjacent tissues (n = 193). b Immunohistochemistry for p-MeCP2(S421), MeCP2 and 5mC in tissue sections selected from cancer samples of low or high ratios in OPNc/OPNa from RT-qPCR measures (n = 15). c , d Quantified staining intensity of p-MeCP2 and MeCP2 levels from ( b ). e Statistics on DNA methylation by 5-mC staining in OPNc/OPNa high or low groups. ** p < 0.01, *** p < 0.001

Article Snippet: The probing antibodies were against the following antigens: MeCP2 (3456, Cell Signaling Technology, Danvers, MA, USA), pMeCP2 (S421) (AP3693a, Abgent, San Diego, CA, USA) and GAPDH (TA-08, ZSGB-BIO, Beijing, China).

Techniques: Expressing, Immunohistochemistry, Quantitative RT-PCR, Staining, DNA Methylation Assay

Journal: Cancer cell

Article Title: The osteogenic niche is a calcium reservoir of bone micrometastases and confers unexpected therapeutic vulnerability

doi: 10.1016/j.ccell.2018.10.002

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: pMeCP2(S421) in 1:1000(WB), Rabbit , Rockland , Cat#600-401-X14, RRID:AB_2614557.

Techniques: Luciferase, Recombinant, Expressing, In Vivo, Software

Journal: Cancer cell

Article Title: The osteogenic niche is a calcium reservoir of bone micrometastases and confers unexpected therapeutic vulnerability

doi: 10.1016/j.ccell.2018.10.002

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Western blots were performed using antibodies against NFAT1(Cell Signalling, 5861S), pS6K(T389) (Cell Signaling, 9234S), pCamKII(T286) (Cell Signalling, 12716S), MeCP2 (Cell Signalling, 3456S), pMeCP2(S421)(Rockland, 600-401-X14), Firefly luciferase (Thermo, PA5-32209).

Techniques: Luciferase, Recombinant, Drug discovery, Expressing, In Vivo, Software