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: Frontiers in Oncology

Article Title: The molecular mechanisms underlying neutrophil infiltration in vessel co-opting colorectal cancer liver metastases

doi: 10.3389/fonc.2022.1004793

Figure Lengend Snippet: The expression of RUNX1 in the cancer cells influences the expression of Ang1 in adjacent hepatocytes in vitro . (A, B) Immunoblotting showing the expression of RUNX1 in colorectal cancer (HT29 and SW620) cells (left panels). The right panels represent the intensity of RUNX1 bands after normalization with GAPDH using ImageJ. Data are from three independent experiments. (C, D) The left panels show the expression of Ang1 in the co-cultured IHH hepatocytes with colorectal cancer (HT29 and SW620) cells. The right panels show the intensity of Ang1 bands after normalization with GAPDH using ImageJ. Data are from three independent experiments. Data are presented as the mean ± SD. ns, Not significant.

Article Snippet: The following primary antibodies were used: RUNX1 1:500 (LS Bio, Seattle WA, USA, #LS-C353932), Ang1 1:1000 (Abcam, Waltham, MA, USA, #ab102015), GAPDH 1:2000 (Abcam, Waltham, MA, USA, #ab9485).

Techniques: Expressing, In Vitro, Western Blot, Cell Culture

Journal: Frontiers in Physiology

Article Title: Multiple Calcium Export Exchangers and Pumps Are a Prominent Feature of Enamel Organ Cells

doi: 10.3389/fphys.2017.00336

Figure Lengend Snippet: Western blot analyses of PMCA1-4, and NCKX3 in secretory-stage and maturation-stage rat enamel organs. (A) Western blot analysis for PMCA1 (Ai) , PMCA2 (Aii) , PMCA3 (Aiii) and PMCA4 (Aiv) . Samples are secretory-stage enamel organ cells (S), maturation-stage enamel organ cells (M), brain tissue (B) and heart tissue (H). Brain and heart samples are shown for comparison, as all PMCAs are highly expressed in brain and at lower levels in the heart (Brini and Carafoli, ; Brini et al., ). Molecular weight markers are indicated at left. The expected molecular weights for PMCA1, PMCA2, PMCA3, and PMCA4 are ~130, 133, 123, and 129 kDa, respectively. The bands are seen for PMCA1, PMCA3, and PMCA4 (boxed and arrow). No expression of PMCA2 is evident. GAPDH is used here as a loading control. (B) Western blot analysis of NCKX3. The expected molecular weight for NCKX3 is ~60 kDa. NCKX3 is expressed in all 4 tissue samples tested, with similar expression noted in both secretory-stage and maturation-stage enamel organ cells, and brain tissue. Relatively higher levels of NCKX3 expression can be appreciated in heart tissue. Amelx and Actc are used as controls as expression is highest in secretory-stage ameloblasts (Lacruz et al., , ) and heart tissue (Hamada et al., ) respectively. GAPDH is used here as a loading control.

Article Snippet: Antibodies against PMCA1 (AbCam, Cambridge, MA, USA; catalog #ab190355), PMCA2 (ab3529), PMCA3 (ab3530), PMCA4 (ab2783), NCKX3 (St. John's Laboratory, London, UK, catalog #STJ94358), GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA, USA, catalog #sc-32233), amelogenin (ThermoFisher Scientific, catalog #PA5-31286), and cardiac muscle actin (ACTC1) (GeneTex Inc., Irvine, CA, catalog #GTX101876) were used at dilutions of 1:500, 1:2,000, 1:300, 1:5,000, 1:500, 1:500, 1:3,000, and 1:500, respectively in 5% milk in TBST.

Techniques: Western Blot, Comparison, Molecular Weight, Expressing, Control

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: Scientific Reports

Article Title: Impact of Escherichia coli K12 and O18:K1 on human platelets: Differential effects on platelet activation, RNAs and proteins

doi: 10.1038/s41598-018-34473-w

Figure Lengend Snippet: Western blot and ELISA analysis of HMBS and ATP2C1. ( a ) Cell lysates were applied to SDS-PAGE gels under reducing conditions. HMBS and ATP2C1 protein were detected in platelets using a polyclonal antibody. Incubation of platelets with E. coli K12 and E. coli O18:K1 in 1:5 or 1:10 platelet-bacteria ratios converted HMBS 47 kDa form to a 40 kDa protein. ATP2C1 was not affected. GAPDH was used as a loading control. ( b ) The releasates of the platelet-bacteria mix were collected after centrifugation (500 g, 10 minutes without break). HMBS, ATP2C1 and LRCH4 levels were measured by ELISA. Data represents the mean of three independent experiments (n = 3). ATP2C1 was detectable in platelet supernatants, while HMBS and LRCH4 proteins were either not released from platelets or in concentrations below the detection level of the ELISA (data not shown). BDL, below detection limit. Western blot results are representative image of three replications. The same exposure was applied equally across the entire image. The original pictures of the full-length western blots can be found in Supplementary Fig. .

Article Snippet: The blots were incubated overnight with primary anti-human antibodies (rabbit polyclonal ATP2C1 (1:750), LRCH4 (1:500), HMBS (1:1000) or GAPDH (1:600) from Atlas Antibodies, Bromma, Sweden).

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, SDS Page, Incubation, Bacteria, Control, Centrifugation