Journal: The American Journal of Pathology

Article Title: MicroRNA 96 Is a Post-Transcriptional Suppressor of Anaplastic Lymphoma Kinase Expression

doi: 10.1016/j.ajpath.2012.01.008

Figure Lengend Snippet: A: A Western blot shows that miR-96 down-regulates the expression of NPM-ALK in Karpas 299 and SUP-M2, EML4-ALK in H2228, and ALKF1174L in SH-SY5Y cell lines. Control cells were transfected with cel-miR-67. The decrease in the different forms of ALK protein was associated with a marked decrease in the corresponding phosphorylated ALK protein, which was also associated with a pronounced down-regulation of pAkt, pSTAT3, pJNK, JunB, and pIGF-IR. Changes were not seen in the basal levels of Akt, STAT3, JNK, or IGF-IR proteins. β-Actin shows equal protein loading. The Western blot studies were repeated four times with consistent results. B: A Western blot shows that siRNA induced a marked decrease in ALK protein levels in Karpas 299, SUP-M2, H2228, and SH-SY5Y cell lines. β-Actin shows equal protein loading. C: The decrease of NPM-ALK by siRNA in Karpas 299 and SUP-M2 cell lines was associated with a significant increase in miR-96 expression (P = 0.001 and P = 0.023, respectively, compared with cells transfected with scrambled siRNA). D and E: The decrease in EML4-ALK in H2228 (D) and ALKF1174L in SH-SY5Y (E) cell lines by ALK siRNA was also associated with a marked increase in miR-96 levels (P = 0.0008 and P = 0.02, respectively). The mean ± SE values of three different experiments, performed in triplicate, are shown.

Article Snippet: Antibodies Antibodies to phosphorylated ALK (pALK, Tyr1604; 3341), pSTAT3 (Tyr705; 9131), pIGF-IR (Tyr1131; 3021), Akt (9272), pAkt (Ser473; 4051), JNK (3708s), pJNK (Thr183/Tyr185; 4668), and JunB (3753) were obtained from Cell Signaling Technology (Danvers, MA).

Techniques: Western Blot, Expressing, Control, Transfection

Journal: Scientific Reports

Article Title: Distribution of insulin in trigeminal nerve and brain after intranasal administration

doi: 10.1038/s41598-019-39191-5

Figure Lengend Snippet: IN administration of insulin increases levels of phospho(Y1185)-insulin receptor in brain. One hour after IN administration of insulin or saline, Western blot analysis with an antibody specific for phospho(Y1185) insulin receptor was performed on whole brain homogenates. Rats administered IN insulin had significantly higher levels of phospho(Y1185)-insulin receptor in brain compared to rats administered IN saline. Relative expression levels are presented as percentage vs. control. * P < 0.02 ( n = 4). The full-length blot is presented in Supplementary Fig. .

Article Snippet: Phospho-insulin receptor beta (Y1185) antibody was purchased from Bioss (#bs-5453R).

Techniques: Western Blot, Expressing

Journal: Aging cell

Article Title: Metallothionein antagonizes aging-induced cardiac contractile dysfunction: role of PTP1B, insulin receptor tyrosine phosphorylation and Akt.

doi: 10.1111/j.1474-9726.2006.00201.x

Figure Lengend Snippet: Fig. 6 Western blot analysis exhibiting insulin receptor, PTP1B and insulin receptor tyrosine phosphorylation (Tyr1146) in ventricles from young or old FVB and metallothionein (MT) mice challenged with or without insulin (5 mU g−1, i.p. for 10 min). (A) Represent blots depicting insulin receptor, PTP1B and insulin receptor tyrosine phosphorylation (Tyr1146) (with or without insulin treatment) using respective antibodies; (B) insulin receptor expression; (C) PTP1B expression; and (D) phosphorylation of insulin receptor (Tyr1146) under basal and insulin-stimulated condition. Mean ± SEM, n = 6–11, *P < 0.05 vs. FVB young-group, **P < 0.05 vs. corresponding non-insulin treated group, #P < 0.05 vs. FVB-old group.

Article Snippet: Anti-Akt, anti-pAkt (Ser473), antiphospho-insulin receptor (Tyr1146) and anti-β-actin antibodies were obtained from Cell Signaling (Beverly, MA, USA).

Techniques: Western Blot, Phospho-proteomics, Expressing

Journal: iScience

Article Title: CD36 regulates diurnal glucose metabolism and hepatic clock to maintain glucose homeostasis in mice

doi: 10.1016/j.isci.2023.106524

Figure Lengend Snippet: Knockdown of CD36 facilitates nuclear FoxO1 and Per1 expression (see also Figure S3 ) (A) Colocalization of CD36 and IRβ was imaged in CD36 flox/flox mouse livers and HepG2 cells. Images show the overlay of IRβ (green) and CD36 (red). Yellow punctate structures indicate CD36-IRβ colocalization. Scale bars = 10μm. (B) Co-IP of CD36 and IRβ in CD36 flox/flox mouse liver tissue and HepG2 cells. IP was performed with anti-IRβ antibody, and immunoblotting was used to detect CD36 and IRβ. (C) Western blot analysis of AKT, p-AKT (Ser473), PI3K p85, and p-PI3K p85 (Try467) expression in HepG2 cells after insulin (100 nmol/L, 5 min) stimulation. The relative quantification is shown on the right. (D) Immunohistochemistry staining of FoxO1 and Per1 in the livers of CD36 flox/flox and CD36-LKO mice. Fifty cells were used for statistical analysis of the number of nuclei positive for FoxO1 and Per1 puncta. Scale bars = 100μm. (E and F) Effect of CD36 knockdown and the addition of SC79 on the cellular localization of FoxO1 (E) and Per1 (F) in HepG2 cells. Fifty cells were used for statistical analysis of nuclei positive for FoxO1 and Per1 puncta. Scale bars = 10μm. (G) Cellular localization of FoxO1 and Per1 was imaged in NIH3T3cells after CD36 knockdown. Fifty cells were used for statistical analysis of nuclei positive for FoxO1 and Per1 puncta. Scale bars = 10μm. (H and I) Immunoblots showing the effect of CD36 knockdown and the addition of SC79 on FoxO1 and Per1 levels in the nucleus (H) and cytoplasm (I) of HepG2 cells. n = 3 in each group. All data are shown as the mean ± SEM. Comparison of different groups was carried out using two-tailed unpaired Student’s t test (D and G) or two-way ANOVA (C, E, F, H and I). ∗p <0.05, ∗∗p <0.01 versus control groups.

Article Snippet: Cells grown in confocal dishes were fixed with 4% paraformaldehyde for 15 min at 37°C, blocked with 3% bovine serum albumin for 30 min at 37°C, and incubated overnight at 4°C with primary antibodies against Per1 (LS-C807611, LifeSpan, US), FoxO1 (2880S, Cell Signaling, US) and IRβ (bs-0290R, Bioss, China).

Techniques: Knockdown, Expressing, Co-Immunoprecipitation Assay, Western Blot, Immunohistochemistry, Staining, Comparison, Two Tailed Test, Control

Journal: iScience

Article Title: CD36 regulates diurnal glucose metabolism and hepatic clock to maintain glucose homeostasis in mice

doi: 10.1016/j.isci.2023.106524

Figure Lengend Snippet:

Article Snippet: Cells grown in confocal dishes were fixed with 4% paraformaldehyde for 15 min at 37°C, blocked with 3% bovine serum albumin for 30 min at 37°C, and incubated overnight at 4°C with primary antibodies against Per1 (LS-C807611, LifeSpan, US), FoxO1 (2880S, Cell Signaling, US) and IRβ (bs-0290R, Bioss, China).

Techniques: Recombinant, Magnetic Beads, Enzyme-linked Immunosorbent Assay, Protein Extraction, Chromatin Immunoprecipitation, Western Blot, Software, Laser-Scanning Microscopy