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: Translational Oncology

Article Title: Co-targeting MCL-1 and ERK1/2 kinase induces mitochondrial apoptosis in rhabdomyosarcoma cells

doi: 10.1016/j.tranon.2021.101313

Figure Lengend Snippet: BIM, BMF and MCL-1 are key players in cell death induction upon Ulixertinib/S63845 co-treatment RMS13 and RH41 cells were transiently transfected with non-silencing siRNA (siCtrl) or three distinct siRNA sequences targeting BIM (siBIM #1, siBIM #2, siBIM #3) or BMF (siBMF #1, siBMF #2, siBMF #3) and treated with 6 μM Ulixertinib and/or 1 μM S63845 (RMS13) and 2 μM Ulixertinib and/or 0.1 μM S63845 (RH41). Expression of BIM and BMF was assessed by Western blotting after 48 h. β-actin was used as loading control. Representative blots of two independent experiments are shown (A, C). Cell death was measured after 72 h by fluorescence microscopy analysis of PI uptake using Hoechst 33342 and PI co-staining (B, D). RMS13 and RH41 cells were transiently transfected with non-silencing siRNA (siCtrl) or two distinct siRNA sequences targeting MCL-1 (siMCL-1 #1, siMCL-1 #2) and treated as in B, D above. Expression of MCL-1 was assessed by Western blotting after 48 h. GAPDH was used as a loading control. Representative blots of two independent experiments are shown (E). Cell death was measured after 72 h using PI andHoechst 33342 co-staining (F). Mean and SD (error bars) of three independent experiments performed in triplicates are shown, *, P < 0.05; ***, P < 0.001.

Article Snippet: Western blot analysis was performed as described previously , using the following antibodies: rabbit anti-BAX (1:1000, Millipore, ABC11), rabbit anti-BAK (1:1000, Millipore, 06–536), mouse anti-BCL-2 (1:1000, BD Biosciences, 610539), rabbit anti-BCL-X L (1:1000, Cell Signaling, 2762S), rabbit anti-BCL-W (1:1000, Cell Signaling, 2724S), rabbit anti-pERK (Thr202/Tyr204) (1:1000, Cell Signaling, 9101S), rabbit anti-PUMA (1:1000, Cell Signaling, 4976S), rabbit anti-BIM (1:1000, Cell Signaling, 2819S), rabbit anti-p90 RSK (Ser380) (1:1000, Cell Signaling, 11989S), rabbit anti-RSK1 (1:1000, Cell Signaling, 8408S), mouse anti-PARP cleaved (1:1000, Cell Signaling, 9546S), rabbit anti-caspase-3 (1:1000, Cell Signaling, 9662S), rabbit anti-caspase-9 (1:1000, Cell Signaling, 9502S) rabbit anti-ERK (1:10000, Sigma, M5670), mouse anti-β-Actin (1:10000, Sigma, A5441), mouse anti-Vinculin (1:10000, Sigma, V9131), mouse anti-MKP-3 (DUSP6) (1:1000, Santa Cruz Biotechnology, sc-377070), rat anti-BMF (1:500, ENZO, ALX-804-343-C100), rabbit anti-MCL-1 (1:1000, ENZO, ADI-AAP-240-F), mouse anti-NOXA (1:1000, ENZO, ALX-804-408), mouse anti-GAPDH (1:5000, HyTest, 5G4cc(-6C5cc)).

Techniques: Transfection, Expressing, Western Blot, Fluorescence, Microscopy, 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 reports

Article Title: Derlin rhomboid pseudoproteases employ substrate engagement and lipid distortion to enable the retrotranslocation of ERAD membrane substrates

doi: 10.1016/j.celrep.2021.109840

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Mouse anti-GAPDH , BIO-RAD , Cat#MCA4740; RRID:AB_2107457.

Techniques: Virus, Recombinant, Mutagenesis, Positive Control, Software