Journal: Redox biology

Article Title: Dysregulation of sphingolipid and cholesterol homeostasis imposes oxidative stress in human spermatozoa.

doi: 10.1016/j.redox.2025.103669

Figure Lengend Snippet: Fig. 2. MβCD increases PI3K phosphorylation, while inhibition of S1PR or NOS impairs MβCD-induced tyrosine phosphorylation. Spermatozoa were incubated for 3.5 h at 37 ◦C under various conditions: untreated (−), treated with 10 % v/v fetal cord serum ultrafiltrate (FCSu), treated with 0.5 mM methyl- β-cyclodextrin (MβCD), and treated with MβCD in combination with (a) 40 μM VPC 23019 (S1PR1/3 inhibitor) or (b) L-NAME (Nitric Oxide Synthase inhibitor). (a) Immunoblotting analysis revealed that VPC 23019 at 40 μM decreased PI3K phosphorylation (P-PI3K) in spermatozoa incubated with 0.5 mM of MβCD. (b) Immunoblotting showed that L-NAME treatment decreased tyrosine phosphorylation (P-Tyr) in sperm samples treated with FCSu and MβCD. The signal intensity for each lane was normalized to the silver-stain optical density value for accurate quantification. α-Tubulin loading controls were performed for direct comparison with the silver stain loading controls. The data represent sperm samples from four different healthy donors (n = 4). Statistical analysis was performed using ANOVA with Tukey’s test: *p ≤0.05, **p ≤0.01, and ***p ≤0.001.

Article Snippet: Rabbit polyclonal anti-phospho-PI3K (P-PI3K) antibodies were purchased from Cell Signaling (#4228) (Beverly, MA, USA).

Techniques: Phospho-proteomics, Inhibition, Incubation, Western Blot, Silver Staining, Comparison

Journal: eLife

Article Title: SIV-specific neutralizing antibody induction following selection of a PI3K drive-attenuated nef variant

doi: 10.7554/elife.88849

Figure Lengend Snippet: Figure 4. Nef-G63E mutation reduces PI3K/mTORC2 binding and pAkt drive. (A) Representative surface expression level histograms of CD3, CD4, major histocompatibility complex class I (MHC-I), CXCR4, and BST-2 in CD4loNef+ subpopulations after wild-type (WT) or Nef-G63E mutant simian immunodeficiency virus (SIV) infection at multiplicity of infection (MOI) 0.1 on HSC-F cells. (B) Left: representative histograms of relative pAkt serine (Ser) 473 levels in p27+ subpopulations after WT or Nef-G63E mutant SIV infection at MOI 0.1 on HSC-F cells. Numbers show pAkt Ser473 mean fluorescence

Article Snippet: After two washes, they were resuspended in 0.5% BSA/PBS for the prevention of experimental procedurerelated loss and stained with mouse anti- SIVmac251 Nef mAb (clone 17, Thermo Fisher Scientific/ Pierce) or mouse IgG1 isotype control mAb (P3.6.2.8.1, Abcam) in combination with either of the following rabbit antibodies: anti- GβL (86B8, CST), anti- mTOR (7C10, CST), polyclonal anti- human PI3K p85 (Merck Millipore), anti- PI3 Kinase p110α (C73F8, CST), or rabbit IgG1 isotype control mAb (DA1E, CST).

Techniques: Mutagenesis, Binding Assay, Expressing, Immunopeptidomics, Virus, Infection, Fluorescence