Journal: Journal of Biomedical Research

Article Title: RAF1 in AgRP neurons involved in the regulation of energy metabolism via the MAPK signaling pathway

doi: 10.7555/JBR.39.20250114

Figure Lengend Snippet: Raf1 regulated MAPK signaling under insulin stimulation. A: Relative mRNA levels of Raf1 , Agrp , and Npy in the hypothalamus of control and AgRP- Raf1 -OE mice fed an NCD ( n = 5–6 mice). B: Western blotting analysis of protein levels of FLAG, MEK1/2, pMEK1/2, ERK1/2, pERK1/2, CREB, and pCREB in the N42 Raf1 -overexpression cells. β-Actin served as the internal control ( n = 3). C: Phosphorylation levels of MEK1/2, ERK1/2, and CREB proteins in the N42 Raf1 -overexpression cells. D: Western blotting analysis of protein levels of RAF1, MEK1/2, pMEK1/2, ERK1/2, pERK1/2, and pCREB in the N42 Raf1 -knockout cells. β-Actin served as the internal control ( n = 3). E: Phosphorylation levels of MEK1/2, ERK1/2, and CREB proteins in the N42 Raf1 -knockout cells. F: Representative IF staining of pCREB in AgRP neurons of control and AgRP- Raf1 -OE mice following 2 mU insulin (icv) stimulation ( n = 3 mice; scale bars, 100 μm). G: Fluorescence intensity quantification of pCREB co-localized with HA/mCherry ( n = 3 mice; AAV-DIO-mCherry, N = 77; AAV-DIO- Raf1 -HA, N = 105). N represents the cell number, and n represents the mouse number. Data are presented as the mean ± standard error of the mean. * P < 0.05, ** P < 0.01, and *** P < 0.001 by unpaired t -tests (A, C, E, and G). Abbreviations: AgRP, agouti-related peptide; CREB, cAMP response element-binding protein; ERK1/2, extracellular signal-regulated kinases 1 and 2; icv, intra-cerebroventricular injection; IF, immunofluorescence; MEK1/2, mitogen-activated protein kinase kinases 1 and 2; NCD, normal chow diet; NPY, neuropeptide Y; OE, overexpression; p-CREB, phospho-CREB; p-ERK1/2, phospho-ERK1/2; p-MEK1/2, phospho-MEK1/2; RAF1, v-raf-leukemia viral oncogene 1.

Article Snippet: The membranes were blocked with 5% non-fat milk and incubated overnight at 4 °C with primary antibodies, including mouse anti-RAF1 mAb (1∶1000, Cat. #66592-1-Ig, Proteintech, Wuhan, China), rabbit anti-ERK1/2 polyclonal antibody (1∶500, Cat. #BS2265, Bioworld, China), rabbit anti-ERK1/2 (phospho-T202/Y204) polyclonal antibody (1∶5000, Cat. #AP0484, Bioworld), rabbit anti-MEK1/2 polyclonal antibody (1∶500, Cat. #BS3599, Bioworld), rabbit anti-MEK1/2 (phospho-S2218/222) polyclonal antibody (1∶500, Cat. #BS4733, Bioworld), rabbit anti-CREB1 polyclonal antibody (1∶1000, Cat. #12208-1-AP, Proteintech), anti-CREB (phospho S133) (1∶1000, Cat. #ab32096, Abcam, Cambridge, UK), and mouse anti-β-actin mAb (1∶5000, Cat. #BS6007M, Bioworld).

Techniques: Control, Western Blot, Over Expression, Phospho-proteomics, Knock-Out, Staining, Fluorescence, Binding Assay, Injection, Immunofluorescence

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: iScience

Article Title: The sperm quality in DIO male mice is linked to the NF-κB signaling and Ppp2ca expression in the hypothalamus

doi: 10.1016/j.isci.2025.112110

Figure Lengend Snippet: RNA-seq coupled with bioinformatic analysis of ARC enriched with KNDy neurons in DIO male mice (A) Schematic illustration showed laser capture microdissection (LCM) of ARC enriched of Kiss1 neurons. (B) Representative fluorescence image showed the area of ARC enriched of Kiss1 neurons and bright-field image of ARC after LCM, scale bar: 100 μm. (C) Principal-component analysis (PCA) analysis of the RNA-seq data from both NCD group and HFD group. (D) Heatmap of differentially expressed genes (DEGs). (E) Distribution of DEGs in the NCD and HFD groups. (F) Volcano map of DEGs. (G) The top 20 enrichment circles of all gene ontology (GO) terms. (H) The pathway-DEGs network, was constructed based on signal transduction pathway and endocrine system pathway. (I) The distribution of 42 candidate DEGs patway sets including endocrine system, nervous system, and signal tranduction. (J) The protein interaction network of 42 candidate DEGs is represented by circles, with larger circles indicating more protein interactions. Red: upregulated genes, green: downregulated genes, gray: non-significant difference expressed genes. (K–M) The downregulated gene set was regulated by cAMP responsive element binding protein 1 (CREB1) (K), Akt kinase (AKT) (L), and RELA (M) in HFD male mice. (N) The upregulated gene set was regulated by inflammatory activation in HFD male mice.

Article Snippet: CREB1 Rabbit Polyclonal Antibody , Proteintech , Cat# 12208-1-AP; RRID: AB_2245417.

Techniques: RNA Sequencing, Laser Capture Microdissection, Fluorescence, Construct, Transduction, Binding Assay, Activation Assay

Journal: iScience

Article Title: The sperm quality in DIO male mice is linked to the NF-κB signaling and Ppp2ca expression in the hypothalamus

doi: 10.1016/j.isci.2025.112110

Figure Lengend Snippet: The elevated expression of PP2Ac and decreased activities of AKT and CREB1 in the hypothalamus were confirmed in both DIO mice and ARC IKKβ CA mice (A) The representative fluorescence images shows the expression of PP2Ac (Red) in ARC of Kiss1-cre::R26R-EYFP mice under NCD and HFD. The white arrow indicates the expression of PP2Ac in Kiss1 neurons, scale bar: 20 μm. (B) The counting statistics of PP2Ac-positive cells were performed for Kiss1 neurons in the NCD and HFD group ( n = 3). (C) Relative quantification of PP2Ac in Kiss1 neurons in the NCD and HFD group ( n = 3). (D) PP2Ac expression in ARC of ARC Control and ARC Ikkβ CA group male mice, The white arrow indicates the expression of PP2Ac in AAV-infected neurons, scale bar: 20 μm. (E and F) Western blot analysis of the protein levels of PP2Ac, AKT, pAKT, CREB1, and pCREB1 in the hypothalamus of NCD mice and HFD mice (E) and ARC Control and ARC Ikkβ CA mice (F). (G–I) Quantification of protein levels of PP2Ac (G), pAKT (H), and pCREB1 (I) in the hypothalamus of NCD and HFD mice and ARC Control and ARC Ikkβ CA mice ( n = 6). Data are presented as mean ± SEM,∗ indicates a significant difference (∗ means p < 0.05, ∗∗ means p < 0.01, ∗∗∗ means p < 0.001), “ns” indicates non-significant difference, Student’s t test.

Article Snippet: CREB1 Rabbit Polyclonal Antibody , Proteintech , Cat# 12208-1-AP; RRID: AB_2245417.

Techniques: Expressing, Fluorescence, Quantitative Proteomics, Control, Infection, Western Blot

Journal: iScience

Article Title: The sperm quality in DIO male mice is linked to the NF-κB signaling and Ppp2ca expression in the hypothalamus

doi: 10.1016/j.isci.2025.112110

Figure Lengend Snippet: The activation of NF-κB signaling or the overexpression of Ppp2ca led to the inhibition of AKT and CREB1 activities (A) Western blot analysis of Ikkβ - HA, PP2Ac, P65, and pP65 in control and Ikkβ CA cells. (B–E) The quantification of protein levels of pP65 (B), PP2Ac (C), pAKT (D), and pCREB1 (E) in control and Ikkβ CA cells ( n = 3). (F and M) Relative mRNA levels of Fos , Fosb , Fosl2 , Egr1 , and Kiss1 in control and Ikkβ CA cells (F), control and Ppp2ca -OE (M) treated cells ( n = 3). (G) Western blot analysis of AKT, pAKT, CREB1, and pCREB1 in control and Ikkβ CA cells, control and Ppp2ca -OE cells and DMSO treated and Artemisinin treated cells. (H–J, and L) The quantification of protein levels of pAKT, pCREB1 in control and Ppp2ca -OE cells (H and I) and DMSO treated and Artemisinin treated cells (J and L) ( n = 3). (K) Western blot analysis of AKT, pAKT, CREB1, and pCREB1 in the Ikkβ CA cells treated with NC, si Ppp2ca , DMSO, and SC79, as well as in Ppp2ca- OE cells treated with DSMO and SC79. (N–P) The quantification of protein levels of pAKT, and pCREB1 in the Ikkβ CA cells , treated with NC and si Ppp2ca (N) ( n = 3), DMSO and SC79 (O) ( n = 3), and in the Ppp2ca -OE cells treated with DSMO and SC79 (P) ( n = 3). Data are presented as mean ± SEM,∗ indicates a significant difference (∗ means p < 0.05, ∗∗ means p < 0.01, ∗∗∗ means p < 0.001, ∗∗∗∗ means p < 0.0001), Student’s t test.

Article Snippet: CREB1 Rabbit Polyclonal Antibody , Proteintech , Cat# 12208-1-AP; RRID: AB_2245417.

Techniques: Activation Assay, Over Expression, Inhibition, Western Blot, Control

Journal: iScience

Article Title: The sperm quality in DIO male mice is linked to the NF-κB signaling and Ppp2ca expression in the hypothalamus

doi: 10.1016/j.isci.2025.112110

Figure Lengend Snippet: Excessive expression of Ppp2ca in the ARC resulted in disturbances to LH pulse patterns and a decline in sperm quality (A) Schematic depiction of stereotaxic injection of AAV-CAG-EGFP and AAV-CAG-EGFP- Ppp2ca viral vectors into the mouse hypothalamus. (B) The ventral view of AAV virus injected mouse brains under fluorescence microscope, scale bar:1,000 μm. (C) Representative immunofluorescence images showed the expression of EGFP and PP2Ac in the ARC of AAV virus injected mice, scale bar: 20 μm; 3V: Third ventricle. (D) Western blot analysis of AKT, pAKT, CREB1, and pCREB1 in the hypothalamus of ARC Control and ARC Ppp2ca mice. (E) The representative LH pulse curves of ARC Control and ARC Ppp2ca mice, # indicates the peak LH concentration. (F) Mean LH, amplitude, peak frequency, peak LH, and basal LH in whole blood of male mice from ARC Control and ARC Ppp2ca mice ( n = 6). (G) Serum T levels of male mice from ARC Control and ARC Ppp2ca mice ( n = 6). (H) Total sperms, motile sperms, progressive sperms, VAP, VCL, VSL, ALH, BCF, LIN, and STR of male mice from ARC Control ( n = 15) and ARC Ppp2ca mice ( n = 13). Data are presented as mean ± SEM, ∗ indicates a significant difference (∗ means p < 0.05, ∗∗ means p < 0.01, ∗∗∗ means p < 0.001, ∗∗∗∗ means p < 0.0001), “ns” indicates non-significant difference, Student’s t test.

Article Snippet: CREB1 Rabbit Polyclonal Antibody , Proteintech , Cat# 12208-1-AP; RRID: AB_2245417.

Techniques: Expressing, Injection, Virus, Fluorescence, Microscopy, Immunofluorescence, Western Blot, Control, Concentration Assay

Journal: iScience

Article Title: The sperm quality in DIO male mice is linked to the NF-κB signaling and Ppp2ca expression in the hypothalamus

doi: 10.1016/j.isci.2025.112110

Figure Lengend Snippet:

Article Snippet: CREB1 Rabbit Polyclonal Antibody , Proteintech , Cat# 12208-1-AP; RRID: AB_2245417.

Techniques: Recombinant, Modification, SYBR Green Assay, Expressing, Virus, Enzyme-linked Immunosorbent Assay, Hormonal Assay, Fluorescence, Staining, Software, RNA Sequencing