Journal: Frontiers in endocrinology

Article Title: Weighted Gene Co-Expression Network Analysis Identifies ANGPTL4 as a Key Regulator in Diabetic Cardiomyopathy via FAK/SIRT3/ROS Pathway in Cardiomyocyte.

doi: 10.3389/fendo.2021.705154

Figure Lengend Snippet: FIGURE 6 | Screening of a novel therapeutic target for DbCM. (A) Relative mRNA expression of hub genes normalized to a GAPDH internal control (n=6 each). Values are expressed relative to control groups. (B, C) Representative Western blots and analysis of ANGPTL4 expression; p-FAK(Y397)/FAK ratio; SIRT3 expression; Ac-SOD2/SOD2 ratio; P67phox expression; Gp91phox expression; P47phox expression; Cleaved caspase-3 expression; Bcl-2 expression; Bax expression. Equal protein loading was confirmed using an anti-GAPDH antibody (n=6 each). (D) Representative images of ANGPTL4 fluorescence in heart sections. Scale bar, 50 mm. (E) Representative images of DHE fluorescence in heart sections. Scale bar, 50 mm. (F) Representative images of apoptotic cardiomyocytes (200×). The apoptotic cells were detected by TUNEL (green), and the nuclei were detected by DAPI (blue). Scale bar, 50 mm. (G) Representative immunohistochemical stainings of ANGPTL4, P47phox, and Cleaved caspase-3 (n=6 each). Scale bars, 50 mm. (H) Quantification of ANGPTL4 fluorescence intensity in heart sections (n=6 each). (I) Quantification of DHE fluorescence intensity in heart sections (n=6). (J) Percentage of TUNEL positive cells. (K–M) Representative immunohistochemical quantification of ANGPTL4, P47phox, and Cleaved caspase-3. Scale bars, 50 mm. Data are shown as mean ± SD (n=6 each); *p < 0.05, **p < 0.01 vs CON group (student t-test). ns represents statically non-significant; IOD, the integral optical density.

Article Snippet: Membranes were blocked for 1 h in 5% bovine albumin (BSA), incubated with primary antibodies against FAK (1:1,000, Cat# 3285S, Cell Signaling Technology), p-FAK (1:1,000, Cat# 8556S, Cell Signaling Technology), cleaved caspase 3 (1:1,000, Cat# 9661S, Cell Signaling Technology), Bax (1:1,000, Cat# 2772S, Cell Signaling Technology), Ac-SOD2 (1:1,000, Cat# AF4360, Affinity), SOD2 (1:1,000, Cat# bs-20668R, Bioss), SIRT3 (1:1,000, Cat# A5718, Abclonal), Bcl-2 (1:1,000, Cat# A0208, Abclonal), NOXA2/P67phox (1:1,000, Cat# A1178, Abclonal), NOX2/gp91phox (1:1,000, Cat# A1636, Abclonal), NCF1/ P47phox (1:1,000, Cat# A1148, Abclonal), ANGPTL4 (1:1,000, Cat#CSB-PA005044, Cusabio), GAPDH (1:5,000, Cat# CSBMA000071M1m, Cusabio), b-actin (1:5,000, Cat#66009-1-Ig, Proteintech), or b-tubulin (1:1,000, Cat#GB11017B, Servicebio) at 4°C overnight.

Techniques: Expressing, Control, Western Blot, TUNEL Assay, Immunohistochemical staining

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Acupuncture Alters Expression of Insulin Signaling Related Molecules and Improves Insulin Resistance in OLETF Rats

doi: 10.1155/2016/9651592

Figure Lengend Snippet: Effects of acupuncture on protein expressions of PI3K-p85, phospho-PKC ζ / λ , and GLUT4 in skeletal muscle of OLETF rats. Protein expression was determined by Western blot. GAPDH was used as an internal control. Data are shown as mean ± SD ( n = 8 each group). ∗ P < 0.05 and ∗∗ P < 0.01 versus SD group; △△ P < 0.01 versus OLETF group.

Article Snippet: Nonspecific binding sites were blocked with 5% milk powder diluted in TBS with 0.05% Tween 20 (TBST) for 60 min. Proteins were detected using the following antibodies: rabbit polyclonal antibody for PI3K-p85 (diluted 1 : 3000; Bios; bs-0128R), rabbit polyclonal antibody for phospho-PKC ζ / λ (diluted 1 : 3000; CST; #9378), rabbit polyclonal antibody for GLUT4 (diluted 1 : 3000; Boster; BA1626), and mouse monoclonal antibody for GAPDH (diluted 1 : 3000; Boster; BM1623).

Techniques: Expressing, Western Blot, Control

Journal: Cell Cycle

Article Title: Exogenous expression of SAMHD1 inhibits proliferation and induces apoptosis in cutaneous T-cell lymphoma-derived HuT78 cells

doi: 10.1080/15384101.2016.1261226

Figure Lengend Snippet: Exogenous SAMHD1 expression inhibits HuT78 cell growth and proliferation. HuT78 cells expressing hemagglutinin (HA)-tagged SAMHD1 were generated via lentiviral vector-mediated transduction. (A) Exogenous expression of SAMHD1 was validated by immunoblotting analysis using antibodies to HA (SAMHD1) and GAPDH (loading control). (B) Total RNA was isolated from HuT78 vector or SAMHD1-expressing cells and qPCR analysis was performed to quantify the relative SAMHD1 mRNA levels. GAPDH mRNA levels were quantified as internal control. (C) HuT78 vector control and SAMHD1 expressing cells were seeded in triplicate in 24-well plates at a density of 1 × 104 cells/well in 1 ml culture media on day 0 and live cells were counted on indicated days via trypan-blue exclusion method. (D) Cell lines were seeded on day 0 in 4 replicates in 96-well plates at density of 1 × 103 cells/well in 100 µl and cell proliferation was determined on indicated days utilizing an MTS-based proliferation assay. All the data presented are representative of 3 or more independent experiments. (C-D) ***, p < 0.0001.

Article Snippet: 65 Immunoblotting was performed using the following antibodies: HA-11 (1:1000, Covance #901501), caspase-8 (1:1000, Cell signaling #9746S), caspase-3 (1:1000, Cell signaling #9662S), PARP (1:1000, Cell Signaling, #9542S), FLIP S/L (1:500, Santa Cruz Biotechnology sc-5276) and GAPDH (1:3000, Bio-Rad #AHP1628).

Techniques: Expressing, Generated, Plasmid Preparation, Transduction, Western Blot, Control, Isolation, Proliferation Assay