threonine 389  (MedChemExpress)

Journal: Aging Cell

Article Title: Age‐associated cholesterol reduction triggers brain insulin resistance by facilitating ligand‐independent receptor activation and pathway desensitization

doi: 10.1111/acel.12932

Figure Lengend Snippet: Ex vivo cholesterol replenishment in hippocampal slices of old mice restores insulin signaling. (a) Addition of cyclodextrin‐cholesterol (referred as MβCD‐Ch or Cholesterol) solution to old mice hippocampal slices induces a 20% increase in membrane cholesterol. (b, c) Cholesterol addition (as in a) reduces hyperactivated Akt and its downstream target p70S6K. (b) Detection of Akt Phospho‐Serine 473; (c) detection of p70S6K Phospho‐Threonine 389. (d) Cholesterol addition to old hippocampal slices reduces Phospho‐Serine 9 GSK3β inhibitory residue. (e) Addition of the cholesterol solution to hippocampal slices of old mice rescues the impairment of insulin‐LTD. Graphic showing insulin‐LTD induction in old mice slices ( n = 7) and old mice slices incubated with MβCD‐Ch ( n = 9). Black box indicates time of insulin application. Gray box indicates the time of MβCD‐Ch mix application. Representative analogue traces below were collected at the indicated time points. (f, g) Cholesterol replenishment decreases insulin resistance marks on IRS‐1 protein. (f) IRS‐1, Phospho‐Serine 632; (g) Phospho‐Serine 307. The value inside the bars indicates the number of independent experiments. Data are represented as mean ± SEM . t test for (a, b, d, g), Wilcoxon test for (c, f), one‐way ANOVA with post hoc Bonferroni's test for (e). The asterisks indicate the p values (* p < 0.05; ** p < 0.01)

Article Snippet: The following antibodies were used for Western blot (WB) and immunoprecipitation (IP): anti‐α‐Tubulin (WB 1:10,000, Abcam, Cambridge, UK ref.: ab7291), anti‐β‐Actin (WB 1:20,000; Sigma‐Aldrich ref.: A5441), anti‐GAPDH (WB 1:20,000; Abcam, ref.: ab8245), anti‐Akt (WB 1:1,000; Cell Signaling, Danver, MA ref.: #9272), anti–Phospho‐Akt Serine 473 (WB 1:1,000, Cell Signaling ref.: #4060), anti‐GSK3 α/β (WB 1:1,000, Invitrogen, Carlsbad, CA ref.: 44–610), anti‐Phospho GSK3 α/β Serine 21/9 (WB 1:1,000; Cell Signaling ref.: #9331), anti‐IGF‐1 Receptor β (WB 1:1,000, IP 1:100; Cell Signaling ref.: #9750), anti‐Phospho IGF‐1 receptor β Tyr1135/116 (WB 1:1,000; Cell Signaling ref.: #3024), anti‐Insulin receptor β (IP 1:100, Santa Cruz, Dallas, TX ref.: sc‐57342), anti‐Insulin receptor β (WB 1:750, Santa Cruz, Dallas, TX ref.: sc‐711), anti‐Phospho insulin receptor β Tyr1150/1151 (WB 1:750; Millipore, Burlington, MA ref.: 04‐299), anti‐IRS‐1 (WB 1:1,000; BD Biosciences, Franklin Lakes, NJ ref.: 611394), anti‐Phospho IRS‐1 Serine 307 (WB 1:750; Abcam ref.: ab4865), anti‐Phospho IRS‐1 Serine 632 (WB 1:200; Cell Signaling ref.: #2388), anti‐Phospho IRS‐1 Serine 1101 (WB 1:1,000; Cell Signaling ref.: #2385), anti‐mTOR (WB 1:1,000; Cell Signaling ref.: #4517), anti‐Phospho mTOR Serine 2448 (WB 1:2,500; Cell Signaling ref.: #5536), anti‐p70S6K (WB 1:1,000; BD Biosciences ref.: 611260), anti‐Phospho p70S6K Threonine 389 (WB 1:750; Cell Signaling ref.: #9206), anti‐IRS‐2 (WB 1:500, Upstate 06‐506), anti‐Vinculin (WB 1:1,000; Millipore ref.: AB6039).

Techniques: Ex Vivo, Membrane, Residue, Incubation

Journal: Aging Cell

Article Title: Age‐associated cholesterol reduction triggers brain insulin resistance by facilitating ligand‐independent receptor activation and pathway desensitization

doi: 10.1111/acel.12932

Figure Lengend Snippet: In vivo pharmacological inhibition of cholesterol loss in old mice restores hippocampal insulin sensitivity and insulin‐LTD. (a, b) Oral administration of the Cyp46A1 inhibitor Voriconazole reduces hippocampal PI3K/Akt and downstream effector p70S6K hyperactivation in old mice. (a) Western blot of Phospho‐Serine 473 activating residue on Akt. (b) Western blot of p70S6K Phospho‐Threonine 389. (c, d) Oral treatment with Voriconazole decreases insulin resistance marks in hippocampal samples of old mice. (c) Western blots for IRS‐1 resistance marks Phospho‐Serine 632; (d) Phospho‐Serine 307. (e) Voriconazole treatment reduces the inhibitory phosphorylation on GSK3β. (f) Graphic showing insulin‐LTD induction in old mice slices ( n = 7) and old mice slices incubated with Voriconazole ( n = 8). Hippocampal slices were incubated with Voriconazole 10 nM for 60 min starting 30 min before insulin stimulus. Black box indicates time of insulin application. Gray box indicates time of Voriconazole application. Representative analogue traces below were collected at the indicated time points. Bar graphs: Numbers inside indicate the number of independent experiments. Data are represented as mean ± SEM. t test for (a, b, c, d, e). One‐way ANOVA with post hoc Bonferroni's test for ( F ). The asterisks indicate the p values (* p < 0.05)

Article Snippet: The following antibodies were used for Western blot (WB) and immunoprecipitation (IP): anti‐α‐Tubulin (WB 1:10,000, Abcam, Cambridge, UK ref.: ab7291), anti‐β‐Actin (WB 1:20,000; Sigma‐Aldrich ref.: A5441), anti‐GAPDH (WB 1:20,000; Abcam, ref.: ab8245), anti‐Akt (WB 1:1,000; Cell Signaling, Danver, MA ref.: #9272), anti–Phospho‐Akt Serine 473 (WB 1:1,000, Cell Signaling ref.: #4060), anti‐GSK3 α/β (WB 1:1,000, Invitrogen, Carlsbad, CA ref.: 44–610), anti‐Phospho GSK3 α/β Serine 21/9 (WB 1:1,000; Cell Signaling ref.: #9331), anti‐IGF‐1 Receptor β (WB 1:1,000, IP 1:100; Cell Signaling ref.: #9750), anti‐Phospho IGF‐1 receptor β Tyr1135/116 (WB 1:1,000; Cell Signaling ref.: #3024), anti‐Insulin receptor β (IP 1:100, Santa Cruz, Dallas, TX ref.: sc‐57342), anti‐Insulin receptor β (WB 1:750, Santa Cruz, Dallas, TX ref.: sc‐711), anti‐Phospho insulin receptor β Tyr1150/1151 (WB 1:750; Millipore, Burlington, MA ref.: 04‐299), anti‐IRS‐1 (WB 1:1,000; BD Biosciences, Franklin Lakes, NJ ref.: 611394), anti‐Phospho IRS‐1 Serine 307 (WB 1:750; Abcam ref.: ab4865), anti‐Phospho IRS‐1 Serine 632 (WB 1:200; Cell Signaling ref.: #2388), anti‐Phospho IRS‐1 Serine 1101 (WB 1:1,000; Cell Signaling ref.: #2385), anti‐mTOR (WB 1:1,000; Cell Signaling ref.: #4517), anti‐Phospho mTOR Serine 2448 (WB 1:2,500; Cell Signaling ref.: #5536), anti‐p70S6K (WB 1:1,000; BD Biosciences ref.: 611260), anti‐Phospho p70S6K Threonine 389 (WB 1:750; Cell Signaling ref.: #9206), anti‐IRS‐2 (WB 1:500, Upstate 06‐506), anti‐Vinculin (WB 1:1,000; Millipore ref.: AB6039).

Techniques: In Vivo, Inhibition, Western Blot, Residue, Phospho-proteomics, Incubation