Journal: iScience

Article Title: Cell-type specific circadian transcription factor BMAL1 roles in excitotoxic hippocampal lesions to enhance neurogenesis

doi: 10.1016/j.isci.2024.108829

Figure Lengend Snippet:

Article Snippet: Phospho-CREB (Ser133) (87G3) Rabbit mAb , Cell Signaling , Cat#14001; RRID: AB_2798359.

Techniques: TUNEL Assay, Quantitative RT-PCR, Software

Journal: The Journal of Neuroscience

Article Title: β-Amyloid Peptide at Sublethal Concentrations Downregulates Brain-Derived Neurotrophic Factor Functions in Cultured Cortical Neurons

doi: 10.1523/JNEUROSCI.5463-03.2004

Figure Lengend Snippet: Pretreatment with sublethal concentrations of Aβ1-42 (5 or 10 μm) for 2 hr decreased the elevation of phosphorylated CREB levels induced by BDNF (50 ng/ml, 10 min). A, Western blot analysis of CREB phosphorylated at Ser133 (P-CREB) and total CREB (T-CREB). Aβ1-42 treatment resulted in a concentration-dependent decrease in the level of P-CREB but, considering all experiments (n = 3), had no significant effect on T-CREB levels. B, Quantification of the effect of pretreatment with 1, 5, or 10 μm Aβ1-42 (A1, A5, A10; A, here and in all other figures, stands for Aβ1-42). Estimates are the mean ± SEM (n = 3) expressed in terms of P-CREB levels obtained in the BDNF-exposed cultures (B, here and in all other figures, stands for BDNF). The effect of Aβ1-42 was significant (*p < 0.05, unpaired Student's t test). C, Pretreatment with 10 μm Aβ1-42 with random amino acid sequence [Aβ(R)] had no significant influence on the BDNF-induced increase of P-CREB levels. D, Immunohistochemical analysis of P-CREB. P-CREB immunoreactivity increased in BDNF-stimulated cultures (b) compared with unstimulated control (a). BDNF-induced increase in P-CREB immunoreactivity was suppressed by Aβ1-42 (5 μm) treatment (c). E, Exposure of the cultures to Aβ1-42 for 2 hr had no effect on P-CREB levels. In three experiments P-CREB levels in the presence of 5 and 10 μm Aβ1-42, respectively, were 104 ± 2 and 105 ± 6% of basal. F, Analysis of CRE-mediated transcriptional activity. Cortical neurons at 3 DIV were transfected with plasmid pCRE-Luc containing CRE sequences and a luciferase reporter gene (see Materials and Methods). Cells transfected with a CMV-luciferase control plasmid served to normalize CRE activity. After 40 hr the cultures were switched to fresh medium and incubated for 1 hr in the presence or the absence of 5 μm Aβ1-42. Transfected cortical cultures were incubated for an additional 9 hr either with or without the addition of 50 ng/ml BDNF, and transcriptional activity was measured by the luciferase assay. Aβ1-42 treatment decreased the BDNF-induced transcriptional activity of CRE. Estimates are the mean ± SEM (n = 3) expressed in terms of BDNF-induced transcriptional activity. The effect of Aβ1-42 was significant (*p < 0.05, unpaired Student's t test).

Article Snippet: Membranes were incubated additionally for 2 hr at room temperature in the presence of the following antibodies as indicated: from Upstate Cell Signaling (Charlottesville, VA), phosphorylated CREB (P-CREB; detects CREB phosphorylated at Ser 133 ; 1:2000), total CREB (T-CREB; 1:2000), Shc (the antibody recognizes all isoforms; 1:1000), PLCγ (1:1000), total TrkB (T-TrkB; 1:1000); from Cell Signaling Technology (Beverly, MA), phosphorylated MAPK/ERK (P-MAPK; detects p44/42 MAPK phosphorylated at Thr 202 and Tyr 204 ; 1:2000), total MAPK (T-MAPK; 1:2000), phosphorylated Raf-1 (P-Raf-1; detects Raf-1 phosphorylated at Ser 388 ; 1:1000), phosphorylated MEK1/2 (P-MEK1/2; detects MEK1/2 phosphorylated at Ser 217/221 ; 1:1000), total MEK1/2 (T-MEK1/2; 1:1000), phosphorylated Elk-1 (P-Elk-1; detects Elk-1 phosphorylated at Ser 383 ; 1:1000), total Elk-1 (T-Elk-1; 1:1000), phosphorylated Akt (P-Akt; detects Akt phosphorylated at Ser 473 ; 1:1000), total Akt (T-Akt; 1:1000), phosphorylated TrkB (P-TrkB; detects TrkB phosphorylated at Tyr 490 ; 1:500).

Techniques: Western Blot, Concentration Assay, Sequencing, Immunohistochemical staining, Activity Assay, Transfection, Plasmid Preparation, Luciferase, Incubation