Journal: PLoS Biology

Article Title: Ablating astrocyte insulin receptors leads to delayed puberty and hypogonadism in mice

doi: 10.1371/journal.pbio.3000189

Figure Lengend Snippet: (A) Western blotting of PGES2 protein expression from FACS-isolated astrocytes were imaged and quantified for IR loxp and IRKO GFAP mice. IR loxp (black bar) and IRKO GFAP (white bar), ( n = 4 per group, 2 brains pooled per lane). Values are expressed as means ± SEM. * P < 0.05 IRKO GFAP versus IR loxp group. The underlying data can be found in . (B–C) Schematic diagram representing the mechanism of astrocyte modulation of HPG axis. FACS, fluorescence-activated cell sorting; HPG, hypothalamic pituitary gonadotropin; IR, insulin receptor; IRKO GFAP , astrocyte-specific insulin receptor deletion; PGES2, prostaglandin E synthase 2.

Article Snippet: The primary antibodies used were as follows: IRβ (Cat. #3025S, Cell signaling); PGES2 (Cat. #bs-2639R, Bioss) [ , ]; and GADPH (Cat.# SC-32233, Santa Cruz Biotechnology).

Techniques: Western Blot, Expressing, Isolation, Fluorescence, FACS

Journal: Scientific Reports

Article Title: Inceptor as a regulator of brain insulin sensitivity

doi: 10.1038/s41598-023-36248-4

Figure Lengend Snippet: 3xTg Inceptor is detectable in the aged mouse brain and reduced in the 3xTg (AD) hypothalamus but not hippocampus or cortex. 40-week-old female mouse hypothalamus tissue was assessed by western blot for Inceptor, Insulin Receptor (IR), and IGF1 Receptor (IGF1R) ( a ), and quantified compared to actin ( b ). Hippocampus and cortex tissue from the same animals was measured for Inceptor ( c ) and quantified compared to actin ( d ). Unpaired t-test was used to determine statistical significance with p < 0.05.

Article Snippet: Insulin receptor antibody (3025S), IGF1R antibody (3027S), ERK antibody (9102L), pERK antibody (9106L), and pAKT antibody (9271S) were purchased from Cell Signaling technology.

Techniques: Western Blot

Journal: Scientific Reports

Article Title: Inceptor as a regulator of brain insulin sensitivity

doi: 10.1038/s41598-023-36248-4

Figure Lengend Snippet: Inceptor is highest in neurons and immunoprecipitated with IR and IGF1R in cortex, but may not significantly change Insulin/IGF signaling following knockdown. ( a ) Inceptor mRNA transcripts measured by cell type using qPCR and quantified relative to tata-binding-protein (TBP) as a control. Cells were cultured from C57BL/6 J mice or MIN6 immortalized line. ( b ) Blots for Inceptor CO-IP, the related IP (IR or IGF1R) plus input sample blots from the pancreas, liver, cortex, and hypothalamus from experiment performed on two animals. ( c ) Knockdown of Inceptor with lentiviral construct #3. Total inceptor knockdown is shown via western blot compared to actin (on one western blot), and a second blot of the same samples shows total levels of pERK, ERK, pAKT, and AKT. Each lane within a group represents a biological replicate (n of 3–4). ( d ) Statistical analysis of western blots. Statistical significance determined with unpaired t-test for Inceptor knockdown, and two-way ANOVA with Tukey for multiple comparisons for pERK/ERK and pAKT/AKT. There was no statistically significant difference in the interaction term (insulin treatment X inceptor knockdown), p = 0.1346. Some blots here and in other figures have been contrast and brightness adjusted for maximum visibility. Full raw images of all blots are available as noted in Data Availability section, below.

Article Snippet: Insulin receptor antibody (3025S), IGF1R antibody (3027S), ERK antibody (9102L), pERK antibody (9106L), and pAKT antibody (9271S) were purchased from Cell Signaling technology.

Techniques: Immunoprecipitation, Knockdown, Binding Assay, Control, Cell Culture, Co-Immunoprecipitation Assay, Construct, Western Blot