foxo1 cell signaling technology  (Cell Signaling Technology Inc)

Journal: The FASEB Journal

Article Title: KCNB1‐Leptin receptor complexes couple electric and endocrine function in the melanocortin neurons of the hypothalamus

doi: 10.1096/fj.202401931r

Figure Lengend Snippet: FIGURE 6 Canonical and non-canonical leptin pathways are altered in NULL. (A) Graphical illustration of LepR signaling. Canonical (JAK2/STAT3) and non-canonical PI3K/Akt/FOXO1 and ERK pathways are indicated. (B) Representative Western blot and densitometric quantification of the long isoform leptin receptor (LepR) in hypothalamic lysates of WT and NULL mice. (C) Representative Western blot of STAT3 phosphorylated at Y705 (pSTAT3) and total STAT3 protein (STAT3) in hypothalamic lysates of WT and NULL mice in absence/presence of leptin. (D) Densitometric quantifications of the fraction of pSTAT3/STAP calculated from Westerns as that in (C). (E) Representative Western blot of phosphorylated FOXO1 at S256 (pFOXO1) and total FOXO1 protein (FOXO1) in hypothalamic lysates of WT and NULL mice in the absence/presence of leptin. (F) Densitometric quantifications of pFOXO1/FOXO1 calculated from Westerns as those in (E). (G) ELISA quantifications of phosphorylated Akt at S473 (pAkt) over total Akt protein (pAkt/Akt) in hypothalamic lysates of WT and NULL mice in the absence/presence of leptin. (H) Representative Western blots of phosphorylated ERK1/2 at T202/T204 (pERK) and total ERK1/2 (ERK) protein in hypothalamic lysates of WT and NULL mice in the absence/presence of leptin and corresponding densitometric quantifications of the fraction of phosphorylated ERK1/2 over total ERK1/2 (pERK/ERK). (I) ELISA quantifications of αMSH in hypothalamic lysates of WT and NULL mice in the absence/presence of leptin. Western blot loading controls were actin and Bradford colorimetric assay. Animals were injected with either vehicle or 1.0 mg/kg body weight leptin and sacrificed 90 min later. *p < .05 (ANOVA with Tukey's post hoc).

Article Snippet: Reagent or resource Source Identifier Antibodies POMC (D3R1U) Rabbit mAb Cell Signaling Technology #23499 c- Fos (9F6) Rabbit mAb Cell Signaling Technology #2250 Stat3 (124H6) Mouse mAb Cell Signaling Technology #9139 Phospho- Stat3 (Tyr705) (M9C6) Mouse mAb Cell Signaling Technology #4113 FoxO1 (C29H4) Rabbit mAb Cell Signaling Technology #2880 Phospho- FoxO1 (Ser256) (E1F7T) Rabbit mAb Cell Signaling Technology #84192 p44/42 MAP kinase (phosphorylated Erk1/2) Cell Signaling Technology Cat# 9101 p44/42 MAPK (Erk1/2) (137F5) Rabbit mAb Cell Signaling Technology Cat# 4695 Integrin beta- 5 (D24A5) Rabbit mAb Cell Signaling Technology Cat# 3629 Integrin beta- 1 antibody Cell Signaling Technology Cat #4706 Alpha MSH polyclonal rabbit Bioss Antibodies bs- 1848R AgRP polyclonal antibody Thermo Fisher PA5- 118934 Leptin receptor polyclonal antibody Thermo Fisher PA1- 053 Leptin receptor recombinant Rabbit monoclonal antibody Thermo Fisher MA5- 32685 (NIH), Grant/Award Number: R01AG060919; National Science Foundation (NSF), Grant/Award Number: 2030348 upon leptin stimulation.

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, Colorimetric Assay, Injection