Journal: Experimental & Molecular Medicine

Article Title: Phosphodiesterase 4D contributes to angiotensin II-induced abdominal aortic aneurysm through smooth muscle cell apoptosis

doi: 10.1038/s12276-022-00815-y

Figure Lengend Snippet: a Immunofluorescence staining of PDE4D and α smooth muscle actin (α-SMA, smooth muscle cell marker) in human non-AAA sections and AAA tissues (6 μm). We obtained similar results from at least three different sets of human tissues in separate experiments. PDE4D: red, α-SMA: green. b Immunofluorescence staining of PDE4D and α-SMA in mouse control sections and AAA tissues (6 μm). We obtained similar results from at least three different sets of mouse samples in separate experiments. PDE4D: red, α-SMA: green. L: lumen. c RT-PCR analysis of Pde4d expression in rat aortic SMCs treated with Ang II (100 nM, 24 h). ** p < 0.0001, Welch’s t test, mean ± SEM, n = 3 separate experiments. d Representative immunoblot analysis of PDE4D protein expression in SMCs treated with Ang II (100 nM, 24 h). e Quantification of PDE4D protein expression by immunoblotting in ( d ) normalized to GAPDH protein (fold change versus control). ** p < 0.01, unpaired Student’s t test, mean ± SEM, n = 3 separate experiments.

Article Snippet: Immunoblotting of the housekeeping protein GAPDH (1:8000, Proteintech, US, Cat#: 60004-1-Ig) was performed to ensure equal protein loading.

Techniques: Immunofluorescence, Staining, Marker, Control, Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot

Journal: Experimental & Molecular Medicine

Article Title: Phosphodiesterase 4D contributes to angiotensin II-induced abdominal aortic aneurysm through smooth muscle cell apoptosis

doi: 10.1038/s12276-022-00815-y

Figure Lengend Snippet: a Representative immunoblot analysis of caspase-3 and cleaved caspase-3 expression in the SMCs treated with PKI (PKA inhibitor, 10 μM, 60 min) and/or Ang II (100 nM, 24 h) and/or PDE4D siRNA (200 nM, 48 h) as indicated. b Quantification of cleaved caspase-3 protein expression by immunoblotting in ( a ) normalized to caspase-3 protein (fold change versus control). ** p < 0.01, *** p < 0.001, **** p < 0.0001, one-way ANOVA with Holm-Sidak’s post hoc test, mean ± SEM, n = 3 separate experiments. c Immunoblot analysis of caspase-3 and cleaved caspase-3 expression in the SMCs treated with ESI-09 (Epac inhibitor, 100 μM, 60 min) and/or Ang II (100 nM, 24 h) and/or PDE4D siRNA (200 nM, 48 h) as indicated. d Quantification of cleaved caspase-3 protein expression by immunoblotting in (c) normalized to caspase-3 protein (fold change versus control). **** p < 0.0001, ns: no significant difference, one-way ANOVA with Holm–Sidak’s post hoc test, mean ± SEM, n = 3 separate experiments. e Representative immunoblot analysis of phospho-Bad (pBad) and Bad expression in the SMCs treated with PKI (PKA inhibitor, 10 μM, 60 min) and/or Ang II (100 nM, 24 h) and/or PDE4D siRNA (200 nM, 48 h) as indicated. f Quantification of pBad expression by immunoblotting in ( e ) normalized to Bad protein (fold change versus control). ** p < 0.01, **** p < 0.0001, one-way ANOVA with Holm-Sidak’s post hoc test, mean ± SEM, n = 3 separate experiments. g Quantification of pBad expression by immunoblotting in ( e ) normalized to GAPDH protein (fold change versus control). ** p < 0.01, *** p < 0.001, one-way ANOVA with Holm–Sidak’s post hoc test, mean ± SEM, n = 3 separate experiments. h Quantification of Bad expression by immunoblotting in ( e ) normalized to GAPDH protein (fold change versus control). ** p < 0.01, *** p < 0.001, **** p < 0.0001, one-way ANOVA with Holm-Sidak’s post hoc test, mean ± SEM, n = 3 separate experiments. i PDE4D expression was significantly increased in Ang II-induced AAA, aggravating SMC apoptosis, which promoted pBad via the cAMP-PKA axis.

Article Snippet: Immunoblotting of the housekeeping protein GAPDH (1:8000, Proteintech, US, Cat#: 60004-1-Ig) was performed to ensure equal protein loading.

Techniques: Western Blot, Expressing, Control

Journal: Journal of Innate Immunity

Article Title: AIF-1 and RNASET2 Play Complementary Roles in the Innate Immune Response of Medicinal Leech

doi: 10.1159/000493804

Figure Lengend Snippet: AIF-1 and RNASET2 Western blot analysis. Proteins extracted from 3 PBS- and LPS-injected leeches and probed with anti- HmAIF-1 (a) and anti-RNASET2 (b) antibodies. The housekeeping protein D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control, and band intensity appeared to be similar in each loaded sample. The anti- HmAIF-1 antibody detected a specific immunoreactive band of about 18 kDa, while two bands of approximately 36 kDa (the extracellular form) and 29 kDa (the intracellular form) were detected by the anti-RNASET2 antibody. c, d The levels of expression were quantified by densitometry using the Image J software package, and the obtained graphs show the level of expression of the two factors. d The graphic is based on the RNASET2 extracellular form. The individual signals from each lane have been cropped from larger digital images, which are available as supplementary information (see www.karger.com/doi/10.1159/000493804 for all supplementary material). Statistical differences were calculated by one-way ANOVA followed by Tukey's post hoc test, and p < 0.05 was considered statistically significant (between PBS and LPS treatments). Means with different letters indicate significant difference between PBS and LPS treatments at different times. Experiments were performed in triplicate, and data represent mean values ± SEM. Statistical analyses were performed using Statistica 7.0 software (StatSoft Inc., Tulsa, OK, USA), and differences were calculated by one-way ANOVA followed by Fisher's post hoc test, and p < 0.05 was considered statistically significant.

Article Snippet: Bands were normalized by using the ImageJ software package ( http://rsbweb.nih.gov/ij/download.html ), and the housekeeping protein D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH), used as an internal reference, was detected with a rabbit polyclonal anti-human GAPDH IgG (Proteintech, Chicago, IL, USA), diluted at 1: 2,000.

Techniques: Western Blot, Injection, Expressing, Software