Journal: Journal of Translational Medicine

Article Title: NeuroD1-USP1-MYCN axis drives tumor progression in neuroblastoma

doi: 10.1186/s12967-026-07844-5

Figure Lengend Snippet: USP1 inhibits K48-linked polyubiquitination of N-Myc and extends its half-life in MYCN-amplified neuroblastoma. ( A ) 293FT cells were transfected with the indicated plasmids. GFP-NC represents the GFP empty vector, GFP-USP1 represents GFP-tagged USP1, Flag-N-Myc represents Flag-tagged N-Myc, HA-Ub represents HA-tagged ubiquitin, HA-Ub-K48 represents the HA-tagged ubiquitin mutant (retaining only lysine 48), and HA-Ub-K63 represents the HA-tagged ubiquitin mutant (retaining only lysine 63). “+” indicates plasmid transfection, while “-” indicates no transfection. After 24 h of transfection, cells were treated with MG-132 (20 µM, 6 h). Total cell protein was collected, and IP was performed, followed by Western blotting to detect the indicated proteins. The input group represents whole-cell lysates. ( B ) 293FT cells were transfected with the indicated plasmids. GFP-NC represents the GFP empty vector, GFP-USP1 represents GFP-tagged USP1, GFP-USP1 (C90S) represents the catalytically inactive USP1 mutant (C90S; cysteine at position 90 mutated to serine), Flag-N-Myc represents Flag-tagged N-Myc, and HA-Ub-K48 represents the HA-tagged ubiquitin mutant (retaining only lysine 48). “+” indicates plasmid transfection, while “-” indicates no transfection. After 24 h of transfection, cells were treated with MG-132 (20 µM, 6 h). Total cell protein was collected, and IP was performed using a Flag antibody, followed by Western blotting to detect the indicated proteins. The input group represents whole-cell lysates. ( C , D ) IMR-32 and BE(2)-M17 cells were transfected with GFP-NC (GFP empty vector) or GFP-USP1 (GFP-tagged USP1). After 48 h of transfection, cells were treated with cycloheximide (CHX, 100 µg/mL). Protein levels of GFP, N-Myc, and GAPDH were analyzed by Western blot at time points 0, 15, 30, 45, and 60 min. The densitometric analysis of N-Myc bands is shown on the right. The integrated density of the N-Myc band was normalized to the corresponding GAPDH band to calculate the “Integrated Density.” The relative integrated density for each time point was expressed as a fold change relative to the 0-minute time point for the GFP-NC or GFP-USP1 groups. Data was presented as mean ± SD. Statistical significance is indicated by * p < 0.05 and ** p < 0.01

Article Snippet: MG-132, Cycloheximide (CHX), and Doxycycline Hydrochloride (DOX) were purchased from Selleck (China).

Techniques: Amplification, Transfection, Plasmid Preparation, Ubiquitin Proteomics, Mutagenesis, Western Blot

Journal: bioRxiv

Article Title: APP metabolism is regulated by p97/VCP through an autophagy and endolysosome-dependent mechanism

doi: 10.64898/2026.02.03.703492

Figure Lengend Snippet: (A) SY5Y-APP695 cells were transfected with negative control scrambled siRNA (siVCP -) or with VCP siRNA (siVCP +) for 72h and then treated with 40µg/mL of cycloheximide (CHX) for the indicated times from 0 to 3 hrs. Western membranes were immunoblotted with the following antibodies: APP-Cter-C17 (APP im. and APP mat.), VCP, and β-actin as a loading control. (B and C) Densitometric analyses and quantification of immature and mature APP levels were expressed as a percentage of the control (Ctrl) on the y-axis, over CHX treatment time on the x-axis. The black dotted lines represent quantification for the scrambled siRNA, while the grey dotted lines represent the siVCP condition (D). VCP siRNA reduces cell surface APP levels in SY5Y-APP695 cells. SY5Y-APP695 cells were transfected with scrambled siRNA (-) or with VCP siRNA (+) for 72h. Cell-surface proteins were chemically biotinylated and isolated with immobilized avidin beads. Flow-through and avidin-bound proteins were resolved by SDS-PAGE, and APP, VCP, and Calnexin (a cytosolic protein) were detected using antibodies against APP (APP Cter-C17), VCP, and calnexin. (E) Densitometric analysis and quantification of total APP levels. The graph indicates the mean ± SD. n=4 *p<0.05, **p<0.0, Unpaired t-test.

Article Snippet: Cycloheximide (CHX) was purchased from Tocris Bioscience.

Techniques: Transfection, Negative Control, Western Blot, Control, Isolation, Avidin-Biotin Assay, SDS Page