p insr igf1r  (Cell Signaling Technology Inc)

Journal: bioRxiv

Article Title: Follicular mural granulosa cells stockpile glycogen to fuel corpus luteum pre-vascularization

doi: 10.1101/2025.01.22.634063

Figure Lengend Snippet: (A) KEGG analysis revealed activated signaling in fGCs post-ovulation/luteinization. (B) Western blotting displayed alterations in insulin receptor activity in fGCs post-stimulation. (C-E) Insulin signaling mediates hCG-induced glucose uptake and glycogen storage in fGCs. (C) Protein levels related to glucose uptake, glycogen synthesis, and glycogenolysis in fGCs were influenced by activation or inhibition of insulin signaling. (D) Flow cytometry measured changes in fGC glucose uptake capacity upon activation or inhibition of insulin signaling. Left: Representative images. Right: Statistical chart; n = 4 independent fGC samples. (E) Impact of activating or inhibiting insulin signaling on fGC glycogen content; scale bar = 100 μm. (F) Bubble plot of the top 10 transcription factors significantly induced by hCG in fGCs. (G) ChIP-seq analysis revealed RUNX1 binding to Insr and Igf1r promoters. Arrows indicate RUNX1 binding peaks. (H) Effects of inhibiting or activating the Ras/Raf/Mek/Erk signaling cascade on RUNX1, glucose uptake-related, and glycogen synthesis-related protein levels in fGCs. (I) PAS staining illustrated the influence of inhibiting or activating Ras/Raf/Mek/Erk on fGC glycogen content; scale bar = 100 μm. (J) EMSA demonstrated RUNX1 binding to Insr and Igf1r promoter sequences. (K) ChIP-qPCR assay for RUNX1 binding to Insr and Igf1r promoters. Input and IgG are positive and negative controls, respectively. UP: qPCR statistical chart; n = 3 independent fGC samples. (L) Dual-luciferase reporter assay identified specific motifs within Insr and Igf1r promoters interacting with RUNX1; n = 3 independent samples. (M) Knockdown of RUNX1 impacted glycogen synthesis-related protein levels and glycogen content in fGCs. Left: Experimental design. Right: Western blotting and PAS staining; scale bar = 100 μm. Statistical significance were determined using one-way ANOVA followed by Tukey’s post hoc test, values were mean ± SD. Significant differences were denoted by *P<0.05, **P<0.01, ****P<0.001,****P<0.0001.

Article Snippet: After transfer, the membrane was blocked with 5% skim milk powder (Nestle, Switzerland) at room temperature, followed by overnight incubation at 4°C with specific primary antibodies: P-PKCα (1:1000 dilution; 9375T, CST, USA), SLC2A1 (1:1000 dilution; 21829-1-AP, Proteintech, USA), GYS1 (1:1000 dilution; 3886T, CST, USA), GSK3B (1:1000 dilution; 12456T, CST, USA), P-GYS1 (1:1000 dilution; 47043T, CST, USA), P-GSK3B (1:1000 dilution; 9323T, CST, USA), PYGB (1:1000 dilution; 12075-1-AP, Proteintech, USA), UGP2 (1:800 dilution; 10391-1-AP, Proteintech, USA), INSR/IGF1R (1:1000 dilution; A21984, Abclonal, China), P-INSR/IGF1R (1:800 dilution; 3024T, CST, USA), RUNX1 (1:1000 dilution; 25315-1-AP, Proteintech, USA), ERK1/2 (1:1000 dilution; A4782, Abclonal, China), P-ERK1/2 (1:1000 dilution; AP0234, Abclonal, China), GAPDH (1:5000 dilution; AC002, Abclonal, China).

Techniques: Western Blot, Activity Assay, Activation Assay, Inhibition, Flow Cytometry, ChIP-sequencing, Binding Assay, Staining, Luciferase, Reporter Assay, Knockdown