chip grade anti creb antibody  (Cell Signaling Technology Inc)

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: The Primary Cilia are Associated with the Axon Initial Segment in Neurons.

doi: 10.1002/advs.202407405

Figure Lengend Snippet: Figure 8. CREB bound with AnkG promoter to regulate AnkG transcription. A-B) The expression level of p-Akt/T-Akt in hippocampus from SSTR3−/−

Article Snippet: Precleared chromatin supernatants were subjected to immunoprecipitation at 4 °C overnight using ChIP-grade antibodies against p-CREB (9198, Cell Signaling Technology).

Techniques: Expressing

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: The Primary Cilia are Associated with the Axon Initial Segment in Neurons.

doi: 10.1002/advs.202407405

Figure Lengend Snippet: Figure 9. CREB expression restored AnkG structure and plasticity in SSTR3−/−neurons. A-B) Neurons in forskolin condition possessed longer AIS and AIS was shorter with 666-15 and CREB siRNA treatments. A. Immunocytochemistry results for AnkG in the SSTR3+/+ neurons in control, forskolin, 666- 15 and CREB siRNA treatments. MAP2 and GFP (green) indicated morphology of neurons, AnkG (red) marked AIS, and DAPI (blue) marked the nuclei. Scale bar, 10 μm. B. Statistical graph of AIS length with different treatments. Left, One-way ANOVA followed by Dunnet post-hoc test was based on data from three independent experiments; F (2, 146) = 55.51; control versus forsklin, p < 0.001; control versus 666-15, p < 0.001. control, n = 53; forskolin, n = 38; 666-15, n = 58. Right, Statistical graph of AIS length with CREB siRNA expression. p < 0.001; control, n = 35; CREB siRNA, n = 41. Unpaired t test, data from 3 independent experiments. The results were represented by mean ± SEM. C-D) AIS length recovered in SSTR3−/−neurons with forskolin treatments. C. Immunocytochemistry results for AnkG in the SSTR3−/−neurons in control and forskolin condition. MAP2 (green) indicated dendrites and soma, AnkG (red) marked AIS, and DAPI (blue) marked the nuclei. Scale bar, 10 μm. D. Statistical graph of AIS length with forskolin treatments. One-way ANOVA followed by Tukey post-hoc test was based on data from three independent experiments; F (2, 97) = 21.69; Control versus forskolin 24 h, p = 0.002; Control versus forskolin 48 h, p < 0.001; forskolin 24 h versus forskolin 48 h, p = 0.005. control, n = 43; forskolin 24 h, n = 33; forskolin 48 h, n = 24. The results were represented by mean ± SEM. E-F) CREB cDNA expression restored AIS length in SSTR3−/−neurons. E. Immunocytochemistry results for AnkG in the SSTR3−/−neurons with control and CREB cDNA. GFP (green) indicated morphology of neurons, AnkG (red) marked AIS, and

Article Snippet: Precleared chromatin supernatants were subjected to immunoprecipitation at 4 °C overnight using ChIP-grade antibodies against p-CREB (9198, Cell Signaling Technology).

Techniques: Expressing, Immunocytochemistry, Control

Journal: Bone Research

Article Title: Inhibition of sympathetic tone via hypothalamic descending pathway propagates glucocorticoid-induced endothelial impairment and osteonecrosis of the femoral head

doi: 10.1038/s41413-024-00371-3

Figure Lengend Snippet: Fig. 6 Sympathetic nerves stimulates endothelial alternation of glycolysis. a Schematic graph of RNA-seq analysis of the sorted femoral head ECs from vehicle- or MPS-treated mice following Adrb2 agonist (clenbuterol) treatment every other day for 1 week. b Heatmap of RNA-seq data showed expression changes encoding glucose metabolism-related genes of the sorted femoral head ECs from vehicle- or MPS-treated mice following Adrb2 agonist (clenbuterol) treatment every other day for 1 week. c Quantitative RT-PCR analysis of Pfkfb3, Pfkl, Pfkp. Eno, and Hk1 genes expression in femoral head ECs from vehicle- or MPS-treated mice following Adrb2 agonist (clenbuterol) treatment every other day for 1 week. d Quantitative RT-PCR analysis of Pfkfb3 gene expression for ECs treated with vehicle, NE, E, NPY, and DA at the concentration of 0.1 μmol/L, respectively. e Quantitative RT-PCR analysis of Pfkfb3 gene expression for femoral head ECs treated with NE for 0–6 h respectively. f, g Representative images of WB and quantitative analysis of PFKFB3 expression for femoral head ECs treated with NE for 0–6 h respectively. h Quantitative RT-PCR analysis of Pfkfb3 gene expression for femoral head ECs treated with vehicle, NE, MPS, or MPS + NE. i, j Representative images of WB and quantitative analysis of PFKFB3 expression for femoral head ECs treated with vehicle, NE, MPS, or MPS + NE. k, l Representative tube formation images and quantification of total loops and total tube length of femoral head ECs under different treatments as indicated. Scale bar: 100 μm. m, n ECAR profile showing glycolytic function and quantification of glycolytic function parameters for femoral head ECs under different treatments as indicated. Vertical lines indicate the time of addition of glucose (10 mmol/L), oligomycin (1 μmol/L), and 2-DG (50 mmol/L). o−s Measurement of glucose uptake, extracellular and intracellular lactate levels, and intracellular G-6-P as well as pyruvate levels for femoral head ECs under different treatments as indicated. t Quantitative analysis of ELISA assay for cAMP in femoral head ECs in response to different treatments as indicated. u Quantitative analysis of PKA activity assay for cell homogenates of femoral head ECs receiving different treatments as indicated. v, w Representative images of WB and quantitative analysis of pCREB/CREB expression for femoral head ECs under different treatments as indicated. All data were presented as means ± SD, n = 6 per group; *P < 0.05. **P < 0.01. ***P < 0.001. Statistical significance was determined by two-tailed Student’s t-test (c). Statistical significance was determined by one-way ANOVA with Bonferroni post hoc test (d, e, g, h, j, n−u, w). Statistical significance was determined by two-way ANOVA with Bonferroni post hoc test (l)

Article Snippet: ChIP-grade antibodies against CREB (1:50, 9198, Cell Signaling Technology), rabbit IgG (negative control), or histone H3 were used to immunoprecipitate the chromatin fragments, which were then incubated with ChIP-grade protein G magnetic beads at 4 °C for 2 h. The eluted DNA was purified and analyzed using qRT-PCR to evaluate the degree of enrichment of the region of the Pfkfb3 gene promoter after rinsing.

Techniques: RNA Sequencing, Expressing, Quantitative RT-PCR, Gene Expression, Concentration Assay, Enzyme-linked Immunosorbent Assay, Activity Assay, Two Tailed Test

Journal: Bone Research

Article Title: Inhibition of sympathetic tone via hypothalamic descending pathway propagates glucocorticoid-induced endothelial impairment and osteonecrosis of the femoral head

doi: 10.1038/s41413-024-00371-3

Figure Lengend Snippet: Fig. 7 Sympathetic neurotransmitter NE-induced endothelial metabolic alternation and angiogenesis-osteogenesis coupling are mediated by Adrb2/cAMP/CREB/Pfkfb3 signaling. a−c Representative images of WB and quantitative analysis of PFKFB3 and pCREB/CREB expression for MPS-treated femoral head ECs under vehicle or NE treatment after knockdown of Adrb2 by si-Adrb2 or CREB inhibition by 666-15. d, e ECAR profile showed glycolytic function and quantification of glycolytic function parameters in MPS-treated femoral head ECs under different treatments as indicated. Vertical lines indicate the time of addition of glucose (10 mmol/L), oligomycin (1 μmol/L), and 2-DG (50 mmol/L). f −j Measurement of glucose uptake, extracellular and intracellular lactate levels, and intracellular G-6-P as well as pyruvate levels for femoral head ECs under different treatments as indicated. k, l Quantitative RT-PCR analysis of pro-angiogenic (Vegfa, Vegfc, Tgfa, and Tgfb2) and pro- osteogenic (Bmp2, Noggin, and Ptn) genes expression for vehicle or 666-15-treated femoral head ECs with MPS and NE cotreatment following activation of cAMP by db-cAMP or Pfkfb3 overexpression by Ad-Pfkfb3. m, n Representative tube formation images and quantification of total loops and total tube length for femoral head ECs under different treatments as indicated. Scale bar: 100 μm. o, p Representative images of ARS staining and quantification of the positively stained areas in BMSCs after EC-CM treatment. Scale bar: 50 μm. q, r ChIP-qRT-PCR analysis revealed CREB antibody (CREB Ab) immune-precipitate Pfkfb3 promoter domain enrichment relative to the input DNA in femoral head ECs under vehicle or NE treatment. Normal rabbit anti-IgG acted as a negative control. Histone H3 antibody (Histone H3 Ab) pulldown for Rpl30 gene enrichment acted as a positive control. All data were presented as means ± SD, n = 6 per group; *P < 0.05. **P < 0.01. ***P < 0.001. Statistical significance was determined by two-way ANOVA with Bonferroni post hoc test (b, c, e−l, n, p). Statistical significance was determined by two-tailed Student’s t-test (r)

Article Snippet: ChIP-grade antibodies against CREB (1:50, 9198, Cell Signaling Technology), rabbit IgG (negative control), or histone H3 were used to immunoprecipitate the chromatin fragments, which were then incubated with ChIP-grade protein G magnetic beads at 4 °C for 2 h. The eluted DNA was purified and analyzed using qRT-PCR to evaluate the degree of enrichment of the region of the Pfkfb3 gene promoter after rinsing.

Techniques: Expressing, Knockdown, Inhibition, Quantitative RT-PCR, Activation Assay, Over Expression, Staining, Negative Control, Positive Control, Two Tailed Test