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