pbs injections  (R&D Systems)

Journal: Investigative and clinical urology

Article Title: Ablation of IGFBP5 expression alleviates neurogenic erectile dysfunction by inducing neurovascular regeneration.

doi: 10.4111/icu.20240325

Figure Lengend Snippet: Fig. 6. Ablation of IGFBP5 expression reduces ROS production in CNI-induced ED mice. (A) Representative images of immunofluorescence stain- ing of corpus cavernosum (CC) tissue for nitrotyrosine (a marker for peroxynitrite generation, green) and hydroethidine (an oxidative fluorescent dye detecting superoxide anions, red) after ICP studies. Scale bar=100 μm. Nuclei are labeled with DAPI (blue). (B, C) The nitrotyrosine- and hy- droethidine-immunopositive areas were quantified using an image analyzer system (n=4, **p<0.01; ***p<0.001). (D) Representative western blot showing p47 phox, iNOS, and IGFBP5 levels in the mouse cavernous tissue after ICP studies. (E-G) Normalized band intensity values for p47 phox, iNOS, and IGFBP5 (n=4, ***p<0.001). Data are presented as mean±standard error of the mean, with the sham operation group set to a relative ratio of 1. IGFBP5, insulin-like growth factor-binding protein 5; ROS, reactive oxygen species; CNI, cavernous nerve injury; ED, erectile dysfunction; PBS, phosphate-buffered saline; shCon, scrambled short hairpin RNA control; shIGFBP5, short hairpin RNA targeting mouse IGFBP5; ICP, intracavernous pressure; DAPI, 4′,6-diamidino-2-phenylindole; ns, not significant; iNOS, inducible nitric oxide synthase.

Article Snippet: These treatments included intracavernous injections of phosphate-buffered saline (PBS) (20 μL/mouse), scrambled control short hairpin RNA (shRNA) (shCon, 5×104 infection units [IFU] in 20 μL), and shRNA targeting mouse IGFBP5 (shIGFBP5) lentiviral particles (shIGFBP5, 5×104 IFU in 20 μL; Santa Cruz Biotechnology).

Techniques: Expressing, Immunofluorescence, Staining, Marker, Labeling, Western Blot, Binding Assay, Saline, shRNA, Control

Journal: Investigative and clinical urology

Article Title: Ablation of IGFBP5 expression alleviates neurogenic erectile dysfunction by inducing neurovascular regeneration.

doi: 10.4111/icu.20240325

Figure Lengend Snippet: Fig. 7. Ablation of IGFBP5 expression modulates PI3K/AKT and JNK/c-Jun signaling and enhances the expression of neurotrophic factors in CNI- induced ED mice. (A) Representative western blot showing levels of p-c-Jun, c-Jun, p-JNK, JNK, p-AKT, AKT, p-PI3K, PI3K, neurotrophic factors (BDNF, NGF, and NT-3), p-IGF1R, IGF1R, Ang-1, and VEGF in the mouse cavernous tissue after ICP studies. (B-K) Normalized band intensity values for the indicated targets (n=4, *p<0.05; **p<0.01; ***p<0.001). Data are presented as mean±standard error of the mean, with the sham opera- tion group set to a relative ratio of 1. IGFBP5, insulin-like growth factor-binding protein 5; CNI, cavernous nerve injury; ED, erectile dysfunction; shCon, scrambled short hairpin RNA control; shIGFBP5, short hairpin RNA targeting mouse IGFBP5; ICP, intracavernous pressure; BDNF, brain-de- rived neurotrophic factor; NGF, nerve growth factor; NT-3, neurotrophin-3; IGF1R, insulin like growth factor 1 receptor; VEGF, vascular endothelial growth factor; ns, not significant.

Article Snippet: These treatments included intracavernous injections of phosphate-buffered saline (PBS) (20 μL/mouse), scrambled control short hairpin RNA (shRNA) (shCon, 5×104 infection units [IFU] in 20 μL), and shRNA targeting mouse IGFBP5 (shIGFBP5) lentiviral particles (shIGFBP5, 5×104 IFU in 20 μL; Santa Cruz Biotechnology).

Techniques: Expressing, Western Blot, Binding Assay, shRNA, Control

Journal: Nature Communications

Article Title: Sphinganine recruits TLR4 adaptors in macrophages and promotes inflammation in murine models of sepsis and melanoma

doi: 10.1038/s41467-024-50341-w

Figure Lengend Snippet: a Histograms and bar graphs showing percentages of CD38+ and Egr2+ BMDM among all BMDM after M1- or M2-like activation, respectively (n = 3 biological replicates). Independent experiments in Supplementary Fig. . b Immunoblot analysis of WT or Lyz2 -cre BMDM after PBS or LPS stimulation. Independent experiments in Supplementary Fig. . c Histograms and bar graph showing percentages of MyD88-expressing BMDM after PBS or LPS stimulation (n = 3 biological replicates). Results are pooled from 3 independent experiments with each 1 pair of mice; each point represents results from individual experiment. Independent experiment in Supplementary Fig. . d Confocal fluorescence microscopy images, showing MyD88 and TLR4 in WT or Lyz2 -cre BMDM after PBS or LPS stimulation. Independent experiments in Supplementary Fig. . e Illustration of the experimental design in ( f – i ). Myris, myristoylation f Confocal fluorescence microcopy showing MyD88 and TLR4 in PBS-/LPS-treated WT or Lyz2 -cre BMDM transduced with MigR1-GFP or MigR1-GFP-MyrisMyD88. Cell sizes were visualized in bar graphs (WT MigR1-GFP: n = 4; Lyz2 -cre MigR1-GFP: n = 6; MigR1-GFP-MyrisMyD88: n = 3 biological replicates). Bar graph data are pooled from 3 individual experiments (more images in Supplementary Fig. ). g Representative flow cytometry dot plots, showing transduction efficiency in LPS-activated WT or Lyz2 -cre BMDM. More data in Supplementary Fig. . h Flow cytometry dot plots and bar graphs, showing percentages of FSC-A high , SSC-A high GFP + LPS-stimulated BMDM (n = 3 biological replicates). Independent experiment in Supplementary Fig. . i Dot plots and bar graphs showing percentages of GFP + CD38 + LPS-stimulated BMDM (n = 3 biological replicates). Independent experiment in Supplementary Fig. . Data are presented as mean ± SD ( a , c , f , h , i ). Statistical comparisons were performed with two-tailed unpaired Student’s t -tests ( a , f ; data points were normally distributed) and one-way ANOVA tests ( c , h , i ; for simultaneous comparisons of more than two groups). 9-week-old female and male ( a – d , f – i ) C57BL/6 mice were used. e Created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license. Source data are provided as a file.

Article Snippet: We used plasma of PBS- or LPS-injected (Sigma-Aldrich, #L2630) mice or supernatant of PBS- or LPS-stimulated (Sigma-Aldrich, #LPS25) BMDM and performed ELISA according to the manufacturer’s instructions (R&D systems, #DY419, #DY2398, #DY406).

Techniques: Activation Assay, Western Blot, Expressing, Fluorescence, Microscopy, Transduction, Flow Cytometry, Two Tailed Test