Journal: Pharmaceuticals

Article Title: PHPB Attenuated Cognitive Impairment in Type 2 Diabetic KK-Ay Mice by Modulating SIRT1/Insulin Signaling Pathway and Inhibiting Generation of AGEs

doi: 10.3390/ph16020305

Figure Lengend Snippet: PHPB modulated SIRT1 deacetylase activity and insulin signaling pathway in cortex and hippocampus of KK-Ay mice. ( A ) The SIRT1 deacetylase activity (fold increase vs. that of C57 mice) was measured by an enzymatic fluorescence method using commercial kits. ( B – F ) Phosphorylation of IRβ Tyr1150/1151 , IRS-1 Tyr859 , and PI3Kp85α or PI3K Tyr458/p55 Tyr199 ; Akt or phosphorylated Akt Ser473 ; and GSK3β or phosphorylated GSK3β Ser9 were determined with corresponding antibodies by western blotting in the cortex and hippocampus of mice treated by PHPB (150 mg/kg) or rosiglitazone (Ros, 5 mg/kg), and densitometric quantification of them, n = 5–8 for each group. The data are presented as means ± SEM. # p < 0.05, ## p < 0.01 versus C57 group; * p < 0.05 versus KK-Ay group.

Article Snippet: Aliquots of tissue lysates (40 μg of protein each) were separated on 7.5–10% SDS-PAGE, electrotransferred to a polyvinylidene difluoride (PVDF) membrane (Bio-Rad Laboratories Co., Ltd., Hercules, CA, USA), blocked with 5% nonfat milk in TBS-Tween buffer for 1.5 h at room temperature, and incubated overnight at 4 °C with the primary antibody, G-6-PD (ab933, Abcam, Cambridge, UK), phosphorylated Tau Ser404 (#20194), phosphorylated GSK3β Ser9 (#5558), GSK3β (#12456), phosphorylated IRS-1 Tyr859 (#3070), Akt (#4685), phosphorylated Akt Ser473 (#4060), PI3Kp85α (#13666), and phosphorylated PI3K Tyr458/p55 Tyr199 (#17366) (Cell Signaling Technology, Inc., Danvers, MA, USA), GLO1 (SC-133144) and phosphorylated-insulin Rβ Tyr 1150/1151 (SC-81500) (Santa Cruz Biotechnology, Inc., CA, USA), β-actin (ab8224, Abcam, Cambridge, UK), and then with horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology, Inc., CA, USA) for 1 h at room temperature.

Techniques: Histone Deacetylase Assay, Activity Assay, Fluorescence, Western Blot

Journal: Experimental and Therapeutic Medicine

Article Title: Effects of RFRP‑3 on an ovariectomized estrogen‑primed rat model and HEC‑1A human endometrial carcinoma cells

doi: 10.3892/etm.2022.11775

Figure Lengend Snippet: Effects of RFRP-3 on the PI3K/AKT/mTOR pathway in HEC-1A cells. HEC-1A cells in the RFRP-3 group were treated with 10,000 ng/ml RFRP-3 for 24 h. (A) The mRNA expression levels of PI3K, AKT and mTOR were measured by reverse transcription-quantitative PCR. (B) The protein expression levels of PI3K, AKT and mTOR, in addition to AKT phosphorylation, were measured using western blot analysis. (C) Semi-quantification of PI3K, AKT and mTOR expression, as well as AKT phosphorylation, presented as relative expression. * P<0.05, ** P<0.01 and *** P<0.001 vs. control. RFRP-3, RFamide-related peptide 3; p, phosphorylated.

Article Snippet: The membranes were then blocked with 5% skimmed milk at room temperature for 2 h, before being incubated with primary antibodies against GAPDH (rabbit; 1:6,000; cat. no. ab9485; Abcam), Kras (rabbit; 1:2,000; cat. no. ab191595; Abcam), Bcl-2 (rabbit; 1:800; cat. no. ab59348; Abcam), Bax (rabbit; 1:800; cat. no. ab32503; Abcam), PI3K (rabbit; 1:500; cat. no. AP0231; Bioworld Technology, Inc.), AKT (rabbit; 1:10,000; cat. no. ab179463; Abcam), phosphorylated (p)-AKT (rabbit; 1:2,000; cat. no. 4060; Cell Signaling Technology, Inc.), mTOR (rabbit; 1:2,000; cat. no. ab32028; Abcam), LC3B (rabbit; 1:2,000; cat. no. ab192890; Abcam), p62 (rabbit; 1:1,000; cat. no. A19700; ABclonal Biotech Co., Ltd.) and GPR147 (also referred to as NPFF-1 receptor; rabbit; 1:5,00; cat. no. bs-12018R; Beijing Bioss Biotechnology Co., Ltd.) at 4˚C overnight.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot

Journal: bioRxiv

Article Title: Apelin-VEGF-C mRNA delivery as therapeutic for the treatment of secondary lymphedema

doi: 10.1101/2023.01.05.522869

Figure Lengend Snippet: A-I. HDLEC were treated in vitro with conditioned media of NIH3T3 cells infected APLN lentivector. A. relative expression of APLN in NIH3T3 evaluated by RT-qPCR on NIH3T3 transduced by APLN lentivector. B. Expression of APLN in conditioned media evaluated by EIA. C. Representatives phospho-AKT/AKT and phosphor-Erk/Erk immunoblots of HDLEC treated with FBS, conditioned medium containing VEGF-C or APLN. D. Graphs represent quantification of phospho/total protein ratio of at least three independent experiments. All graphical data are mean ± s.e.m. *P < 0.05, twoway ANOVA. E. Representative images of scratch wound healing assay on HDLEC stimulated by VEGF-C or APLN. F. Quantification of migration (***p < 0.01). G. F-actin and VE-Cadherin immunostaining of HDLEC after APLN treatment reveals no effect on lymphatic endothelial monolayer junctions. H. Representatives phospho-eNOS/eNOS immunoblots of HDLEC treated with FBS, conditioned medium containing VEGF-C or APLN. I. Graphs represent quantification of phospho/total protein ratio of at least three independent experiments (*P < 0.05).

Article Snippet: The primary antibodies used are the following: APLN (Genetex GTX37465), E2F8 ((Abcam, AB109596), Total Akt (Santa Cruz biotechnology sc-81434), Phosphorylated Akt (Cell Signaling 4060), total eNOS (Cell Signaling 9572), phosphorylated eNOS (Cell Signaling 9571), Actin (Santa Cruz biotechnology sc-47778).

Techniques: In Vitro, Infection, Expressing, Quantitative RT-PCR, Western Blot, Wound Healing Assay, Migration, Immunostaining