Journal: Acta Biochimica et Biophysica Sinica

Article Title: Modulation of ferroptosis via YY1-SLC7A11 axis in hepatic ischemia-reperfusion injury pathogenesis

doi: 10.3724/abbs.2025093

Figure Lengend Snippet: Regulatory effect of NEDD4L expression in IRI on the ubiquitination-induced degradation of YY1 (A) Influence of MG-132 and leupeptin treatments on the reduction in YY1 expression induced by IRI-OGD/R, as detected by western blot analysis. (B) Detection of YY1 ubiquitination levels after IRI-OGD/R treatment. (C) Intersection of predicted human and mouse upstream E3 ligases of YY1, Venn diagram on the left, and the PPI network of 8 intersecting E3 ligases on the right (interaction score = 0.15). (D,E) Expression levels of 8 E3 ligases in mouse liver tissues and the AML12 cell line after IRI detected by RT-qPCR. (F) Immunohistochemical analysis of NEDD4L expression in mouse liver tissues after IRI-I/R treatment, representative images on the left, and quantitative statistical results on the right (scale bar = 50 μm). (G) Expressions of NEDD4L in mouse liver tissues and the AML12 cell line after IRI detected by western blot analysis. (H) Detection of YY1 expression and ubiquitination levels in AML12 cells overexpressing NEDD4L. (I) Co-IP experiment was used to detect the interaction between YY1 and NEDD4L in oe-NEDD4L. (J) Western blot analysis of the effects of NEDD4L knockdown on YY1 ubiquitination and expression after IRI-OGD/R treatment. (K) Flow cytometry analysis of the effects of NEDD4L knockdown on cell apoptosis induced by IRI-OGD/R; n = 10 per group in animal experiments. Cell experiments were repeated three times. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: For the experiments involving drug treatments, AML12 cells were treated with MG-132 (10 μM; MCE) for 4 h to inhibit proteasome activity and with leupeptin (50 μM; MCE) for 4 h to block protein degradation via the lysosomal pathway .

Techniques: Expressing, Ubiquitin Proteomics, Western Blot, Quantitative RT-PCR, Immunohistochemical staining, Co-Immunoprecipitation Assay, Knockdown, Flow Cytometry

Journal: Molecular cell

Article Title: Methyl-Metabolite Depletion Elicits Adaptive Responses to Support Heterochromatin Stability and Epigenetic Persistence

doi: 10.1016/j.molcel.2020.03.004

Figure Lengend Snippet: Data and Software Availability

Article Snippet: Leupeptin Hemisulfate , Thermo Fisher , Cat#AAJ61188MC.

Techniques: Software, Recombinant, Sequencing, Modification, Extraction, Reverse Transcription, Multiplex sample analysis, Plasmid Preparation, SYBR Green Assay, Staining, cDNA Synthesis, DNA Purification, Purification, Control, Targeted Proteomics

Journal: Molecular cell

Article Title: Methyl-Metabolite Depletion Elicits Adaptive Responses to Support Heterochromatin Stability and Epigenetic Persistence

doi: 10.1016/j.molcel.2020.03.004

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Leupeptin Hemisulfate , Thermo Fisher , Cat#AAJ61188MC.

Techniques: Recombinant, Sequencing, Modification, Extraction, Reverse Transcription, Multiplex sample analysis, Plasmid Preparation, SYBR Green Assay, Staining, cDNA Synthesis, DNA Purification, Purification, Control, Software, Targeted Proteomics

Journal: The Journal of biological chemistry

Article Title: NPC1 plays a role in the trafficking of specific cargo to melanosomes.

doi: 10.1016/j.jbc.2023.105024

Figure Lengend Snippet: Figure 5. NPC1 impairs TYR processing and degradation. A, cells were untreated or treated with bafilomycin A (100 nM, 5 h) and analyzed by Western blotting for TYR N-glycan processing after Endo H and PNGase F glycosidase treatment of cell lysates. Relative expression levels of PNGase F-sensitive TYR and the ratio of Endo H-resistant to Endo H-sensitive TYR were plotted. Quantitative representation as mean ± SD of relative protein expressions and the ratio of Endo H resistant to Endo H sensitive bands (one-way ANOVA analysis; *p < 0.05, **p < 0.01, n = 3 biological replicates). B, visual comparison of cell pellets of MNT-WT and NPC1-KO cells untreated or treated with bafilomycin A1 (100 nM, 5 h). C, immunoblot analysis for TYR, LAMP-2, and CNX in cell lysates and in the enrichment of secreted extracellular vesicles from culture supernatant. D, effect of MG132 (50 μM, 5 h) or inhibitor mix (INH = 14.5 uM leupeptin and 106 uM pepstatin, 5 h) cell treatment on TYR expression, analyzed by Western blotting and densitometry, represented as fold change relative to cells treated with DMSO. Quantitative representation as mean ± SD of relative protein expression, n = 3 biological replicates (one-way ANOVA analysis; *p < 0.05). E, CHX treated MNT-WT and NPC1-KO cells analyzed by immunoblotting for TYR normalized to tubulin and graphic representation of TYR degradation rate as a percentage of control (CHX, 0 h) (n = 2 biological replicates). CHX, cycloheximide; CNX, calnexin; DMSO, dimethyl sulfoxide; Endo H, endoglycosidase H; LAMP, lysosome-associated membrane protein; NPC1, Niemann–Pick type C1; PNGase F, peptide-N glycosidase F; TYR, tyrosinase.

Article Snippet: MG132 (sc-201260), leupeptin hemisulfate (sc295358) and pepstatin A (sc-45036) were from Santa Cruz Biotechnology.

Techniques: Western Blot, Glycoproteomics, Expressing, Comparison, Control, Membrane