Journal: NPJ Systems Biology and Applications

Article Title: Mapping the dynamics of insulin-responsive pathways in the blood–brain barrier endothelium using time-series transcriptomics data

doi: 10.1038/s41540-022-00235-8

Figure Lengend Snippet: a , b Scatter plot of cells’ calcein/EthD-1 fluorescence showing the percentage of total counted cells within the live and dead gates in a untreated control and b insulin-treated (300 minutes) cells. c Immunoblots showing IR-β expression in hCMEC/D3 cells treated with insulin for 40 and 300 minutes. d Dynamic changes of insulin receptor gene (INSR) expression in hCMEC/D3 cells treated with insulin up to 300 minutes.

Article Snippet: The proteins were then electroblotted onto a 0.45 μm nitrocellulose membrane, blocked with 5% nonfat dry milk protein (Bio-Rad Laboratories, Hercules, CA), and incubated overnight at 4 °C with primary antibodies against IR-β (1:1000, #3025, Cell Signaling Technology, Denvers, MA) and GAPDH (1:1000, #5174, Cell Signaling Technology, Danvers, MA).

Techniques: Fluorescence, Control, Western Blot, Expressing

Journal: bioRxiv

Article Title: The hepatocyte insulin receptor is required to program rhythmic gene expression and the liver clock

doi: 10.1101/2021.02.05.430014

Figure Lengend Snippet: A , PCR analysis of IR (insulin receptor) floxed (IR hep+/+ ) and TTR- Tam -Cre (TTR- Tam -Cre +/− ) genes from mice that are liver wild type (WT), (IR hep+/+ ) or liver knockout (IR hep−/− ) for IR using DNA extracted from different organs (n=3 mice per group). B , Relative mRNA expression levels of IR from liver, epididymal white adipose tissue (eWAT), and brown adipose tissue (BAT) samples from IR hep+/+ and IR hep−/− mice (n=3 mice per group). Data represent mean ± sem; statistical analysis was performed using two-way ANOVA with Sidak’s multiple comparison test. *p<0.05; **p<0.01; ***p<0.001 C , Insulin receptor-β was assessed by western blotting in liver, eWAT, and BAT (blots are representative of three independent experiments). D,E , Glucose (2g/kg) tolerance test after 6 h of fasting ( D ) and area under the curve (AUC) corresponding to the glucose tolerance test ( E ) (n=10 mice per group). Data represent mean ± sem; statistical significance was calculated using a two-tailed Student t-test with Bonferroni’s multiple comparisons. F , Quantification of circulating Insulin levels by ELISA (n=6 mice per group). Data represent mean ± sem; statistical analysis was performed using two-way ANOVA with Sidak’s multiple comparison test. # represents significant time effect and * represents significant genotype effect #,*p<0.05; ##,**p<0.01; ###,***p<0.001. G , Quantification of circulating glucose levels (n=6 mice per group). H,I , Insulin receptor-β, AKT phosphorylation, and total AKT were assessed by western blotting in liver ( H ) and relative quantification of AKT phosphorylation over total AKT ( I ) (blots are representative of five independent experiments. Data represent mean ± sem. Statistical analysis was performed using linear regression analysis fitting a 24 h cosinor model. A=amplitude; M=mesor.

Article Snippet: Proteins (20-30 μg) were separated by SDS-PAGE (polyacrylamide gel electrophoresis) and transferred onto nitrocellulose membranes, which were first incubated overnight at 4°C with primary antibodies against Insulin receptor β (sc-57342, Santa Cruz Biotechnology, Santa Cruz, CA, USA), pSer473AKT (#4058, Cell Signaling Technology, Beverly, MA, USA), total AKT (#9272, Cell Signaling Technology, Beverly, MA, USA) and β-actin (#4970, Cell Signaling Technology, Beverly, MA, USA), and then with horseradish peroxidase-conjugated secondary antibodies (anti-mouse for Insulin receptor β detection and anti-rabbit for all others) for 1 h at room temperature.

Techniques: Knock-Out, Expressing, Comparison, Western Blot, Two Tailed Test, Enzyme-linked Immunosorbent Assay, Phospho-proteomics, Quantitative Proteomics

Journal: Diabetes

Article Title: Persistent Insulin Resistance in Podocytes Caused by Epigenetic Changes of SHP-1 in Diabetes.

doi: 10.2337/db16-0254

Figure Lengend Snippet: Figure 7: Continuous inhibition of the insulin-stimulated IR-β phosphorylation in podocytes exposed to high

Article Snippet: Reagents and antibodies – Primary antibodies for immunoblotting and immunochemistry were obtained from commercial sources, including actin (horseradish peroxidase; I-19), SHP-1 (C-19), SHP1 (D11; immunofluorescence), IR-β (C-19), phospho-tyrosine (PY99), phosphatase and tensin homolog (PTEN, C-20-R), TGF-β (SC-146), PKC-δ (C-20), cadherin (C-19) and WT-1 (C-19) and rabbit and 5 mouse peroxidase-conjugated secondary antibody from Santa Cruz Biotechnology; protein kinase B (Akt), extracellular signal-regulated kinase (ERK), phospho-Akt (D9E), and phospho-ERK (D13.14.4E) from Cell Signaling; purified monoclonal antibody against PTP1B (610139) and SHP-2 (610621) from BD Biosciences; monoclonal antibody against IR-β (AB69508) from Abcam; nephrin GP-N2 (Progen), podocin (P-0372; Sigma), collagen type IV (Novus Biological), Alexa-546conjugated anti-rabbit and Alexa Fluor 488-conjugated anti-guinea pig from Jackson ImmunoResearch Laboratories, and Alexa Fluor 594-conjugated anti-mouse from Invitrogen.

Techniques: Inhibition, Phospho-proteomics