Journal: bioRxiv

Article Title: In vivo kinetics of protein degradation by individual proteasomes

doi: 10.64898/2026.01.19.700426

Figure Lengend Snippet: a, Cumulative incidence curve showing fraction of SERPs subjected to N-to-C degradation in the presence or absence p97 inhibitor CB5083. Control data is replotted from for comparison (n = 130 traces (control) and n = 70 traces (+CB5083) from 2-3 independent repeats). b, Proteasomal degradation rate for N-to-C degradation in the presence or absence of CB5083. Each data point represents the median from an independent experiment. Horizontal line and error bars represent mean ± s.d of medians (n = 62 traces (control) and n = 36 traces (+CB5083) from 3 independent repeats). Unpaired Student’s t-test was used for statistical analysis. c, Kaplan-Meier survival curves showing proteasomal processivity for N-to-C degradation in the presence or absence of CB5083. Line and shaded region indicate mean ± s.d (n = 62 traces (control) and n = 36 traces (+CB5083) from 2-3 independent repeats). Control data is replotted from . d, Data fitting approach for the trace shown in to identify the onset of ribosome stalling and the onset of protein degradation. (a,c), Log-rank (Mantel-Cox) test was used for statistical analysis.

Article Snippet: To examine the impact of proteasome and p97/VCP inhibition on SERP degradation, cells were pre-treated for 10 minutes with either MG132 (10 μM; Merck) or CB5083 (5 μM; MedChemExpress) prior to the start of imaging.

Techniques: Control, Comparison

Journal: bioRxiv

Article Title: In vivo kinetics of protein degradation by individual proteasomes

doi: 10.64898/2026.01.19.700426

Figure Lengend Snippet: a, Analysis of proteasomal degradation rate for C-to-N directionality in the presence or absence of the p97 inhibitor CB5083. Each data point represents the median from an independent experiment. Horizontal line and error bars represent mean ± s.d of medians (n = 63 traces (control) and 95 traces (+CB5083) from 3 independent repeats). b, Kaplan-Meier survival curves representing proteasome processivity for C-to-N degradation in the presence or absence of CB5083. Line and shaded region indicate mean ± s.d (n = 63 traces (control) and 95 traces (+CB5083) from 3 independent repeats). c, Schematic illustrating the role of p97 in extracting the nascent polypeptide from the ribosome to allow proteasome engagement with the nascent polypeptide. d,e, Representative intensity-time trace for a SERP undergoing synthesis and subsequent decay upon ribosome quality control. The plateau phase represents the time from ribosome stalling at the end of the cleaved mRNA to degradation by the proteasome. Untreated cell (d) and cell treated with the p97 inhibitor CB5083 (e) are shown. The pronounced plateau phase upon p97 inhibition is likely caused by slow extraction of the nascent chain from the 60S ribosome. f, Cumulative incidence curve showing the fraction of SERPs that has initiated degradation relative to the moment of ribosome stalling (i.e. plateau onset) (n = 167 traces (control) and 95 traces (+CB5083) from 3-6 independent repeats). g, Schematic illustrating internal substrate engagement by the proteasome. h, Schematic of the socRNA used for internal substrate recruitment experiments. The socRNA encodes 15×SunTag repeats, 2×ALFA-tag repeats, and a single dTAG-inducible degron. i, Cells expressing the internal dTAG SERP shown in (h) were treated with 500 nM dTAG ligand (dTAGv-1) in the presence or absence of the p97 inhibitor CB5083. Scale bars, 10 μm. j, Quantification of SERP degradation kinetics following addition of 500 nM dTAGv-1. Pre-treatment of cells with p97 inhibitor CB5083 prevents degradation. Line and shaded region indicate mean ± s.d (n = 10 fields of view (control), n = 8 fields of view (+dTAGv-1) and n = 7 fields of view (+dTAGv-1+CB5083) from 2-3 independent repeats). k, Design of socRNA used to assess internal degradation via poly-leucine degrons, which is made up of two stretches of five consecutive leucine residues. l, Quantification of degradation kinetics of polyleucine-containing SERPs encoded by the socRNA construct shown in (k). Pre-treatment of cells with VCP inhibitor CB5083 prevents degradation. Line and shaded region indicate mean ± s.d (n = 10 fields of view (control), n = 8 fields of view (2x(Leu) 5 ) and n = 8 fields of view (2x(Leu) 5 +CB5083) from 2-3 independent repeats). m, Fraction of SERPs undergoing fragmentation during the degradation phase across indicated assays. Each data point represents the median from an independent experiment (n = 41 traces (N-to-C assay), n = 55 traces (C-to-N assay) and n = 44 traces (5 nM dTAGv-1) from 2 independent repeats). n, Average SERP size aligned to the moment of protein fragmentation in cells treated with 5 nM dTAGv-1. Dashed line represents the moment of SERP fragmentation. SERPs were produced as shown in (h). Only events that exhibited fragmentation were included in the analysis. Line and shaded region indicate mean ± s.d (n = 23 traces from 2 independent repeats). (a, m), Unpaired Student’s t-test was used for statistical analysis. * denotes p < 0.05. (b, f), Log-rank (Mantel-Cox) test was used for statistical analysis. **** denotes p<0.0001.

Article Snippet: To examine the impact of proteasome and p97/VCP inhibition on SERP degradation, cells were pre-treated for 10 minutes with either MG132 (10 μM; Merck) or CB5083 (5 μM; MedChemExpress) prior to the start of imaging.

Techniques: Control, Inhibition, Extraction, Expressing, Construct, Produced

Journal: bioRxiv

Article Title: In vivo kinetics of protein degradation by individual proteasomes

doi: 10.64898/2026.01.19.700426

Figure Lengend Snippet: a, Schematic of the socRNA used for substrate-internal proteasome recruitment experiments. The socRNA encodes 15× SunTag repeats, 2× ALFA-tag repeats, a single dTAG-inducible degron and an unstructured region of 60, 120 or 180 amino acids in size. b, Quantification of SERP degradation kinetics following addition of 500 nM dTAGv-1. Lines and shaded region indicate mean ± s.d (n = 8 fields of view (no insert control), n = 7 fields of view (no insert +CB5083), n = 7 fields of view (60 aa unstructured region +CB5083), n = 6 fields of view (120 aa unstructured region +CB5083) and n = 8 fields of view (180 aa unstructured region +CB5083) from 2-3 independent repeats). Dotted lines representing control SERP are replotted from . c, Quantification of SERP degradation kinetics following addition of varying concentrations of dTAGv-1. SERPs were produced from socRNA shown in . Lines and shaded region indicate mean ± s.d (n = 7 fields of view (no drug control), n = 6 fields of view (500 pM dTAGv-1), n = 7 fields of view (5 nM dTAGv-1), n = 6 fields of view (50 nM dTAGv-1) and n = 6 fields of view (500 nM dTAGv-1) from 2 independent repeats). d, Schematic depicting fragmentation of SERP as a consequence of substrate-internal proteasome recruitment. Upon fragmentation, one of the two SERP fragments is degraded, while the other fragment remains stable over time. e, Representative intensity time-trace of a SERP undergoing fragmentation and degradation in the presence of 5 nM dTAGv-1. One of the two SERP fragments is completely degraded while the other SERP fragment remains stable over time. f, Quantification of number of SERP fragments that underwent degradation after fragmentation into two fragments. Bars represent the average of 2 independent repeats, individual experiments are shown as dots (n = 22 traces from 2 independent repeats). g, Rate of SERP degradation induced by 5 nM dTAGv-1. Data points represent individual degradation events. Horizontal line and error bars represent mean ± s.d (n = 40 traces from 2 independent repeats).

Article Snippet: To examine the impact of proteasome and p97/VCP inhibition on SERP degradation, cells were pre-treated for 10 minutes with either MG132 (10 μM; Merck) or CB5083 (5 μM; MedChemExpress) prior to the start of imaging.

Techniques: Control, Produced

Journal: bioRxiv

Article Title: Minute-scale control of ubiquitin-mediated degradation reveals dynamics of bacterial secreted effector-functions

doi: 10.1101/2025.11.19.688170

Figure Lengend Snippet: (A) Immunofluorescence microscopy confirms Cdu1 depletion dynamics. Cdu1 (green, anti-FLAG), chlamydial inclusion (Hsp60,red), and DNA (DAPI, blue). Scale bar = 10 µm. (B) Expansion microscopy (4× expansion) resolving Cdu1 localization patterns under undegraded and degraded conditions. Scale bar = 10 µm. (C) Cdu1 degradation depends on ubiquitin-proteasome and p97 pathways. Host cells were pretreated with inhibitors for 3 hours (including 1 hour during 5-Ph-IAA treatment) with pan-E1 (TAK-243, 1 µM), proteasome (MG-132, 10 µM; bortezomib, 1 µM), or p97 (CB-5083, 10 µM) inhibitors. Degradation was blocked despite 1-hour 5-Ph-IAA exposure. (D) Quantification of inhibitor effects on Cdu1 degradation. FLAG levels (normalized to OmpA, mean ± SD) from three independent immunoblot replicates (one representative in C). Significance assessed by one-way ANOVA with Tukey Multiple comparisons test (*p < 0.05; n.s., not significant). (E) Immunofluorescence validation of inhibitors. Inclusion-localized Cdu1 signal persists in treated cultures. Scale bar = 10 µm. All experiments were replicated ≥3 times with consistent results.

Article Snippet: Proteasome inhibitors (MG-132 at 10 μM, bortezomib at 1 μM; Cell Signaling), p97 inhibitor CB-5083 (10 μM; MedChemExpress), and ubiquitin-activating enzyme inhibitor TAK-243 (1 μM; TAK-243) were prepared as 1,000× stock solutions in DMSO.

Techniques: Immunofluorescence, Microscopy, Ubiquitin Proteomics, Western Blot, Biomarker Discovery

Journal: Nature Communications

Article Title: DAF-16/FOXO and HLH-30/TFEB comprise a cooperative regulatory axis controlling tubular lysosome induction in C. elegans

doi: 10.1038/s41467-025-64832-x

Figure Lengend Snippet: a Endogenously tagged SPIN-1::mCherry in starved L1 worms with and without dSVIP overexpression. Scale bar, 5 µm. b Quantification of lysosomal junctions per object for the genotypes indicated. WT ( N = 20 worms), WT + dSVIP , daf-16(mu86) , daf-16(mu86) + dSVIP , hlh-30(tm1978) and hlh-30(tm1978) + dSVIP ( N = 30 worms per genotype) , daf-16(mu86); hlh-30(tm1978) and daf-16(mu86); hlh-30(tm1978) + dSVIP ( N = 20 worms per genotype). Mean ± s.e.m. One-way ANOVA with Šídák’s multiple comparisons test. (ns = not significant, ** p < 0.01, **** p < 0.0001) ( c ) Endogenously tagged SPIN-1::mCherry in fed day 1 adult worms with and without dSVIP overexpression. Scale bar, 5 µm. d Quantification of lysosomal junctions per object for the genotypes indicated. WT ( N = 28 worms), WT + dSVIP ( N = 20 worms), daf-16(mu86) ( N = 28 worms), daf-16(mu86) + dSVIP ( N = 28 worms), hlh-30(tm1978) ( N = 29 worms), hlh-30(tm1978) + dSVIP ( N = 25 worms) , daf-16(mu86); hlh-30(tm1978) ( N = 28 worms), daf-16(mu86); hlh-30(tm1978) + dSVIP ( N = 29 worms). Mean ± s.e.m. One-way ANOVA with Šídák’s multiple comparisons test. (ns = not significant, **** p < 0.0001). e Endogenously tagged SPIN-1::mCherry in fed WT ( N2 ) and daf-16(mu86) worms with gut dSVIP OE at day 1 of adulthood that were fed control DMSO or CB5083 VCP inhibitor beginning at L4 larval stage. Scale bar, 5 µm. f Quantification of lysosomal junctions per object for the genotypes and conditions indicated. WT ( N2 ) + DMSO ( N = 19 worms), daf-16(mu86); gut dSVIP OE + DMSO ( N = 18 worms), WT ( N2 ) + CB5083 ( N = 15 worms), daf-16(mu86); gut dSVIP OE + CB5083 ( N = 15 worms). Mean ± s.e.m. One-way ANOVA with Šídák’s multiple comparisons test. (**** p < 0.0001). g Endogenously tagged SPIN-1::mCherry in day 7 WT ( N2 ) animals that were fed DMSO or CB5083 VCP inhibitor beginning at day 5 of adulthood. Scale bar, 5 µm. h Quantification of lysosomal junctions per object. ( N = 20 control worms and N = 16 fed CB5083 worms). Mean ± s.e.m. Unpaired two-tailed Student’s t -test. (ns = not significant). i Endogenously tagged SPIN-1::mCherry in day 1 eat-2(ad1116) adults that were fed DMSO or CB5083 VCP inhibitor beginning at L4 larval stage. Scale bar, 5 µm. j Quantification of lysosomal junctions per object. eat-2(ad1116) +DMSO ( N = 20 worms), eat-2(ad1116) +CB5083 ( N = 16 worms). Mean ± s.e.m. Unpaired two-tailed Student’s t -test. (ns = not significant). For all experiments, data were pooled from two independent experiments.

Article Snippet: A 10 μM stock solution of the VCP inhibitor CB5083 (MedChem Express, Cat. # HY-12861/CS-5405) was prepared in DMSO and diluted to a final working concentration of 1 μM in M9 buffer.

Techniques: Over Expression, Control, Two Tailed Test