Journal: Molecular medicine reports

Article Title: σ-1 receptor stimulation protects against pressure-induced damage through InsR-MAPK signaling in human trabecular meshwork cells.

doi: 10.3892/mmr.2017.6647

Figure Lengend Snippet: Figure 6. Expression of Sig‑1R, InsR and p/tERK1/2 protein in hTMCs treated with (+)PTZ and BD‑1063. (A) Western blotting: The molecular sizes of the Sig‑1R, InsR and GAPDH bands of 80 mmHg, 80 mmHg + (+)PTZ, and 80 mmHg + BD‑1063 + (+)PTZ groups are indicated. (B) Western blotting: The molecular sizes of the pERK1/2 and tERK1/2 bands of 80 mmHg, 80 mmHg + (+)PTZ, and 80 mmHg + BD‑1063 + (+)PTZ groups are indicated. (C) Data from densitometric scans of blots from (A and B); Sig‑1R and InsR proteins were increased simultaneously when (+)PTZ was administered, and this effect was attenuated by BD‑1063. Data are presented as the mean ± standard deviation. *P=0.0002 and #P=0.0039 vs. controls (80 mmHg); **P=0.0002 and ##P=0.0039 vs. 80 mmHg + (+)PTZ group. p/tERK1/2 was increased when (+)PTZ was administered and such effect was also attenuated by BD‑1063. &P=0.0137 vs. control (0 mmHg); &&P=0.0137 vs. 80 mmHg + (+)PTZ group. Data are presented as the mean ± standard deviation. Sig‑1R, σ‑1 receptor; InsR, insulin receptor; p/t, phosphorylated and total; ERK, extracellular signal‑regulated kinase; hTMCs, human trabecular meshwork cells; PTZ, pentazocin; BD‑1063, N‑(2‑(3,4‑dichlorophenyl)ethyl)‑N‑methyl‑2‑(dimethylamino) ethylamine Sig‑1R, σ‑1 receptor; InsR, insulin receptor.

Article Snippet: Membranes were blocked with 5% skimmed milk for 1 h, incubated with anti-Sig-1R monoclonal antibody (cat. no. sc-166392; 1:100), anti-InsR polyclonal antibody (cat. no. sc-710; 1:200), anti-pERK polyclonal antibody (cat. no. sc-16982R; 1:200), anti-tERK monoclonal antibody (cat. no. sc-514302; 1:200; all Santa Cruz Biotechnology Inc., Dallas, TX, USA) and anti-GAPDH polyclonal antibody (cat. no. KC-5G4; 1:1,000; Kangcheng Biotechnology Co., Ltd., Shanghai, China) respectively at 4 ̊C overnight, and GAPDH was used as an internal control.

Techniques: Expressing, Western Blot, Standard Deviation, Control

Journal: Scientific Reports

Article Title: High glucose induces trafficking of prorenin receptor and stimulates profibrotic factors in the collecting duct

doi: 10.1038/s41598-021-93296-4

Figure Lengend Snippet: High glucose increases ERK pathway and fibrotic factors via PRR in M-1 cells. ( A ) M-1 cells were transfected with a green fluorescent protein (GFP)-shRNA PRR GFP or an empty plasmid as a control. ( B ) Representative immunoblot showing that shRNA PRR reduced PRR protein levels by 83% as compared to cells transfected with an empty plasmid (n = 7). ( C ) Representative immunoblots and densitometric analysis of TGF β, collagen 1α and fibronectin protein levels after 6 h of incubation with normal (NG), high glucose (HG) and HG conditions plus PRR knockdown (n = 4). No differences in TGF-β, collagen 1α and fibronectin protein levels were observed in NG conditions in cells transfected with shRNA-PRR as compared to controls (data not shown). The β-actin was used as a housekeeping protein. ( D ). Phospho-ERK (P-ERK) intensity versus total ERK (T-ERK) represented as fold change of control demonstrated the increased ratio between P-ERK and T-ERK in HG conditions (10 min) that was partially prevented by shRNA PRR transfections (n = 4). *P < 0.05 versus NG group.

Article Snippet: Activation of ERK pathway was determined by using mouse anti-phospho-p44/42 ERK1/2 (Thr202/Tyr204) antibody (Cat. # 91065, CELL SIGNALING TECHNOLOGY, Beverly, MA), and a rabbit anti total ERK (Cat. # 9122, CELL SIGNALING TECHNOLOGY, Beverly, MA).

Techniques: Transfection, shRNA, Plasmid Preparation, Western Blot, Incubation

Journal: Neuropharmacology

Article Title: FTY720-Mitoxy Reduces Toxicity Associated with α-Synuclein and Oxidative Stress by Increasing Trophic Factor Expression and Myelin Protein in OLN-93 Oligodendroglia Cell Cultures

doi: 10.1016/j.neuropharm.2019.107701

Figure Lengend Snippet: (A) Representative immunoblots and histograms of data from repeated assays measuring AcH3 and total H3 levels in cells treated with vehicle, 160 nM FTY720, 160 nM FTY720-Mitoxy, or 160 nM FTY720-C2 for 24 hr. Only FTY720-Mitoxy significantly increases AcH3 levels. (B) Representative immunoblots and histogram of data from repeated assays measuring pERK1/2 and total ERK1/2 in cells treated with vehicle, 160 nM FTY720, 160 nM FTY720-Mitoxy and 160 nM FTY720-C2 for 24 hr. pERK1/2 level increases were observed after FTY720-Mitoxy treatment. Data represent the mean ± SEM of 3 experiments. One-way ANOVA, * p < .05.

Article Snippet: Antibodies included AcH3 (Lys9/Lys14) (Cat 9677, Cell Signaling Technology, Inc., Danvers, MA, USA; 1:500), β-Actin (Cat 13E5, Cell Signaling Technology Inc.; 1:500), total histone H3 (Cat 96C10, Cell Signaling Technology Inc.; 1:500), phosphorylated ERK1/2 (Tyr204) (Cat sc-7383, Santa Cruz Biotechnology Inc.; 1:200), and total ERK1/2 (Cat sc-93, Santa Cruz Biotechnology Inc.; 1:200), MAG (Cat sc-15324, H-300, Santa Cruz Biotechnology Inc.; 1:200).

Techniques: Western Blot