rabbit polyclonal antibody against phospho creb ser133  (Cell Signaling Technology Inc)

Journal: bioRxiv

Article Title: Structural basis and physiological significance of non-canonical Gs coupling to the prototypical Gi-coupled melatonin MT 1 receptor

doi: 10.1101/2025.11.08.687348

Figure Lengend Snippet: (a-f) Melatonin MT 1 receptor activated G s -cAMP pathway in HEK293T cells. (a) (b) Concentration–response effect of melatonin on forskolin (FSK)-induced cAMP production (30 min, FSK [2 µM], IBMX [1 mM]) in HEK293T cells expressing MT 1 -WT or MT 2 -WT, assessed by HTRF assay without (a) or with (b) PTX treatment (400 ng/mL, 4 h). Data represent means ± SEM of at least 4 independent experiments. (c-f) NanoBiT complementation assay showing recruitment of LgBiT–miniG i (c,e) or LgBiT–miniG s (d,f) to human MT 1 -NP (c, d) or MT 2 -NP (e, f) upon melatonin stimulation (1 µM, 2 min). Data are presented as means ± SEM of five independent experiments. Statistical significance was determined using paired two-tailed t-tests, exact p-values are reported. (g-j) Melatonin (MLT) induces cAMP production in mouse pituitary pars tuberalis (PT) ex vivo . (g) MLT (10 µM) were exposed to sliced PT / mediobasal hypothalamus (MBH) tissue complex of B6 mice kept under 12L/12D conditions. (h) Sixteen-hour exposure of MLT significantly increased cAMP levels in the PT/MBH compared with control 0.1 % DMSO exposure group. Data are means ± SEM of 4 independent experiments. (i) Western blot analysis of phosphorylate CREB (pCREB [Ser133]) in cultured PT/MBH tissues. MLT significantly increased pCREB. Data are means ± SEM of 4 independent experiments. Unpaired t-tests with Welch’s correction were used and one-tailed p-values are reported. (k-m) MLT induces cAMP production in mouse pituitary PT in vivo . (k) MLT (0.26 mM / 0.1 mL) was injected intraperitoneally (i.p.) at ZT8 to B6 mice kept under 8L/16D conditions. After 16 h, brains were extracted and sectioned coronally for immunohistochemistry. ZT, Zeitgeber time; ZT1 indicates light onset time. (l) Representative images of pCREB immunoreactivity (ir) in the PT and MBH of MLT-injected mice. 3V, Third ventricle. (m) Relative changes of pCREB-ir cells normalized by area in the PT. MLT injection increased pCREB (** p < 0.01, t-test). Data are means ± SEM of 6-7 independent experiments. Unpaired t-tests with Welch’s correction were used and one-tailed p-values are reported.

Article Snippet: After blocking with normal rabbit serum (Vectastain), sections were incubated with rabbit polyclonal antibody against phospho-CREB (Ser133) (1:500; #9198, Cell Signaling Technology) at 4°C for 48 h. After encapsulation with mounting medium, images were detected using a BZ-X800 (Keyence).

Techniques: Concentration Assay, Expressing, HTRF Assay, Two Tailed Test, Ex Vivo, Control, Western Blot, Cell Culture, One-tailed Test, In Vivo, Injection, Immunohistochemistry

Journal: Experimental and Therapeutic Medicine

Article Title: Estrogen administration attenuates post-stroke depression by enhancing CREB/BDNF/TrkB signaling in the rat hippocampus

doi: 10.3892/etm.2021.9850

Figure Lengend Snippet: Effects of estrogen treatment on pCREB/CREB and pTrkB/TrkB expression in the hippocampus of PSD rats. (A) Immunolabeling for pCREB in the hippocampal DG region. (Aa) Control group; (Ab) PSD group; (Ac) E2 group. (B) Immunolabeling for CREB in hippocampus. (C) Western blot analysis detected the expected size protein band of pCREB and CREB. (D) Western blot analysis for pTrkB and TrkB in hippocampus in different groups. (E) Quantification of pCREB cells in the hippocampal DG region. (F) Quantitative analysis of western blot images shown in (C). (G) Quantitative analysis of western blot images presented in (D). Data are presented as the mean ± SEM (n=6 animals). * P<0.05; ** P<0.01. CREB, cAMP response element-binding protein; PSD, post-stroke depression; DG, dentategyru; p, phosphorylated; E2, 17β-estradiol; TrkB, tyrosine kinase B; OD, optical density.

Article Snippet: The sections were blocked in 3% H 2 O 2 for 10 min at room temperature, and 3% normal goat serum (OriGene Technologies, Inc.), and then incubated with rabbit polyclonal antibodies against phospho cAMP response element-binding protein (pCREB; 1:800; cat. no. 9198; Cell Signaling Technology, Inc.), CREB (1:3,000; cat. no. 9197; Cell Signaling Technology, Inc.), BDNF (1:200; cat. no. ab6201; Abcam) at 4 ̊C overnight.

Techniques: Expressing, Immunolabeling, Control, Western Blot, Binding Assay

Journal: Experimental and Therapeutic Medicine

Article Title: Estrogen administration attenuates post-stroke depression by enhancing CREB/BDNF/TrkB signaling in the rat hippocampus

doi: 10.3892/etm.2021.9850

Figure Lengend Snippet: Effects of estrogen treatment on subventricle zone infusions of the MAPK kinase (MEK) inhibitor U0126 on pCREB. (A) Protein levels after U0126, aCSF or vehicle (DMSO) treatment, respectively. (B) Densitometric analysis of western blot analysis shown in (A). (C) Western blot analysis of BDNF and β-actin in hippocampus in K252a, aCSF and vehicle (DMSO) groups. (D) Quantitative analysis of western blot analysis. Values are presented as the mean ± SEM (n=6). * P<0.05. CREB, cAMP response element-binding protein; aCSF, artificial cerebrospinal fluid; BDNF, brain-derived neurotrophic factor; p, phosphorylated; OD, optical density.

Article Snippet: The sections were blocked in 3% H 2 O 2 for 10 min at room temperature, and 3% normal goat serum (OriGene Technologies, Inc.), and then incubated with rabbit polyclonal antibodies against phospho cAMP response element-binding protein (pCREB; 1:800; cat. no. 9198; Cell Signaling Technology, Inc.), CREB (1:3,000; cat. no. 9197; Cell Signaling Technology, Inc.), BDNF (1:200; cat. no. ab6201; Abcam) at 4 ̊C overnight.

Techniques: Western Blot, Binding Assay, Derivative Assay

Journal: Disease Models & Mechanisms

Article Title: Schwann cells are activated by ATP released from neurons in an in vitro cellular model of Miller Fisher syndrome

doi: 10.1242/dmm.027870

Figure Lengend Snippet: Neuronal ATP induces CREB phosphorylation in Schwann cells in co-culture with neurons exposed to anti-GQ1b antibody plus complement. (A) Immunofluorescence of SCMN and SC co-cultures exposed for 10 min to saline, FS3+NHS, NHS, FS3+HI-NHS or FS3+NHS plus apyrase. SCs are S100-positive cells (green), phospho-CREB is revealed by a specific antibody (red), nuclei are stained by Hoechst (blue). Scale bars: 10 µm. (B) Quantification of phospho-CREB in S100-positive cells. *** P <0.001; N =5. (C) Representative western blot showing phospho-CREB levels in SCMN and SC co-cultures exposed to FS3+NHS for 10, 15 and 30 min (as indicated), and their reduction by apyrase. At 10 min incubation, phospho-CREB signal is significantly higher in FS3+NHS samples with respect to untreated co-cultures. ATP 100 µM is used here as positive control. No phospho-CREB is detectable in neurons exposed to FS3+NHS, demonstrating that phospho-CREB signal in co-culture lysates derives from SCs. (D) Quantification of western blot in C. Data are normalized to the housekeeping protein Hsp90 and expressed as percentage of the untreated control. * P <0.05; N =5 (Student's t -test, unpaired, two-tailed).

Article Snippet: After saturation, membranes were incubated overnight with a rabbit polyclonal antibody against phospho-CREB (p-CREB) (1:1000, Cell Signaling, 9198S), followed by a secondary anti-rabbit secondary HRP-conjugated antibody (Calbiochem, 1:2000, 401393).

Techniques: Phospho-proteomics, Co-Culture Assay, Immunofluorescence, Saline, Staining, Western Blot, Incubation, Positive Control, Control, Two Tailed Test