Journal: microPublication Biology

Article Title: Turnover of missense mutant cytosolic proteins proceeds in the absence of major quality control E3 ligases

doi: 10.17912/micropub.biology.001990

Figure Lengend Snippet: A) Predominant subcellular localization of the QC E3 ligases and reporters used in this study. Dark and light blue represent strong or circumstantial localization evidence in the indicated compartment, respectively, while white indicates no evidence. Data for subcellular localization of E3 ligases was determined through Human Protein Atlas, Uniprot annotations, and existing literature. STUB1, UBE2O, and UBR4 have been reported to localize to both the cytosol and nucleus, whereas UBR5 and HUWE1 are primarily nuclear (Go et al., 2021; Kim et al., 2018; Mashtalir et al., 2014; Tasaki et al., 2013). Subcellular localization of the reporters was determined by microscopy in a previous study (Baker et al., 2025). B) Western blots of indicated E3 ligase KO cells along with the parental HEK293T cells (HEK). * = indicates unspecific band. HUWE1 KO clones were a gift from Yihong Ye’s lab (Xu et al., 2016). C) Schematic of bicistronic fluorescent reporter construct used in D). D) Log 2 (EGFP/DsRed) levels of the missense mutant reporters and corresponding WT in STUB1 KO cells. Three independent KO clones of STUB1 were used. Results from a one-way ANOVA test are shown with the mean and standard deviation (n=3-9, p > 0.05 = ns). E) Schematic of bicistronic fluorescent reporter used in F-J. F) Log 2 (mClover2/RFP) of the mutant reporter and corresponding WT in HEK293T cells. Results from an unpaired t-test are shown with the mean and standard deviation (n=3; **** p < 0.0001). G-I) Log 2 (mClover2/RFP) of the missense mutant reporters CBS L456P, GNMT H177N, and TPMT A80P expressed in (G) UBR4, (H) UBR5 and (I) HUWE1 WT and KO clone(s). Results from an unpaired t-test are shown with the mean and standard deviation (n=3; p > 0.05 = ns, * p < 0.05, ** p <0.01, *** p < 0.001). J) Density plot of the Log 2 (mClover2/RFP) signal of CBS L456P, GNMT H177N, and TPMT A80P in UBE2O WT and KO clones (n=1).

Article Snippet: Primary antibodies used: STUB1 (Cell Signalling 2080S, 1:1000 dilution), UBE2O (Millipore Sigma HPA023605 1:1000), UBR4 (abcam ab86738, 1:1000), UBR5 (Cell Signalling D608Z, 1:1000), HUWE1 (Bethyl A300-486A, 1:1000), g-Tubulin (Sigma T6557, 1:5000), GAPDH (UBC AbLab Mouse, 1:10,000).

Techniques: Mutagenesis, Microscopy, Western Blot, Clone Assay, Construct, Standard Deviation

Journal: Journal of Virology

Article Title: ALDH1L1 suppresses the replication of porcine epidemic diarrhea virus by degrading viral nucleocapsid and envelope proteins

doi: 10.1128/jvi.01933-25

Figure Lengend Snippet: ALDH1L1 inhibits PEDV replication via the E3 ubiquitin ligase STUB1. ( A ) After HEK 293T cells were cotransfected with plasmids encoding HA-STUB1 and Flag-ALDH1L1, the Co-IP assay was performed using anti-Flag-bound beads. ( B ) After HEK 293T cells were transfected with plasmids coding Flag-ALDH1L1, Co-IP assays were performed using an anti-Flag antibody. ( C ) After inducing GST-STUB1 and ALDH1L1 expression in the BL21(DE3) strain, the interaction between ALDH1L1 and STUB1 was examined via the GST pull-down assay. ( D ) After cotransfecting HeLa cells with Flag-ALDH1L1 and STUB1-HA for 24 h via incubation with an anti-Flag Mab, ALDH1L1 and STUB1 colocalization was confirmed via confocal immunofluorescence microscopy. Scale bars = 100 µm. ( E and F ) After cotransfection with Myc-ALDH1L1, HA-Ub, HA-STUB1, and Flag-N, or Flag-E, HEK 293T cells were treated with CQ and collected. The ubiquitinated N and E proteins were subjected to western blotting after immunoprecipitation with an anti-Flag antibody. ( G and H ) After cotransfecting HEK 293T cells with siRNAs (negative control or STUBA siRNA) and HA-N or HA-E and Flag-ALDH1L1-coding plasmids, western blotting was performed with an anti-HA antibody.

Article Snippet: Anti-ACTB/β-actin antibody (66009-1-Ig), anti-GST antibody (HRP-66001), anti-STUB1 antibody (68407-1-Ig), anti-TOLLIP antibody (68170-1-Ig), horseradish peroxidase-labeled anti-rabbit and mouse antibodies, and IgG antibodies (SA00001-1, SA00001-2) were supplied by Proteintech.

Techniques: Ubiquitin Proteomics, Co-Immunoprecipitation Assay, Transfection, Expressing, Pull Down Assay, Incubation, Immunofluorescence, Microscopy, Cotransfection, Western Blot, Immunoprecipitation, Negative Control

Journal: Journal of Virology

Article Title: ALDH1L1 suppresses the replication of porcine epidemic diarrhea virus by degrading viral nucleocapsid and envelope proteins

doi: 10.1128/jvi.01933-25

Figure Lengend Snippet: Antiviral mechanism by which ALDH1L1 inhibits PEDV replication. During infection with PEDV, ALDH1L1 interacts with the E3 ubiquitin ligase STUB1 to ubiquitinate the PEDV N or E protein. Subsequently, it recruits the cargo receptor TOLLIP to recognize and autophagosomally transport ubiquitinated N or E protein for degradation.

Article Snippet: Anti-ACTB/β-actin antibody (66009-1-Ig), anti-GST antibody (HRP-66001), anti-STUB1 antibody (68407-1-Ig), anti-TOLLIP antibody (68170-1-Ig), horseradish peroxidase-labeled anti-rabbit and mouse antibodies, and IgG antibodies (SA00001-1, SA00001-2) were supplied by Proteintech.

Techniques: Infection, Ubiquitin Proteomics

Journal: European Journal of Medical Research

Article Title: STUB1-mediated PGC-1α ubiquitination promotes postoperative cognitive dysfunction in aged mice via modulating autophagy–lysosome machinery

doi: 10.1186/s40001-025-03771-1

Figure Lengend Snippet: Aberrant STUB1/PGC-1α/TFEB axis in the hippocampus of aged mice following anesthesia and surgery. A , B RNA-seq for screening differentially expressed transcripts in the hippocampus of aged mice following anesthesia and surgery. The screening criteria were |log2fold change|> 1 and adjusted p < 0.05. C – G Significant enrichment of ( C ) apoptosis, ( D ) mitophagy, ( E ) lysosome, ( F ) ubiquitin-mediated proteolysis, and ( G ) proteasome pathways by the differentially expressed transcripts. H – J qRT-PCR of Stub1 , Pgc-1α , and Tfeb transcript levels in the hippocampus of POCD and control mice. K , L Multiple immunofluorescence of STUB1 and PGC-1α in the hippocampus of POCD and control mice. M , N Multiple immunofluorescence of PGC-1α and TFEB in the hippocampus of POCD and control mice. Scale bar, 50 μm; n = 6 per group. ** p < 0.01; *** p < 0.001; **** p < 0.0001 (Student’s t test)

Article Snippet: The membrane was rinsed by TBST (3 times × 5 min), and then slowly shaken with 5% BSA at 37 °C for 2 h. Incubation with primary antibodies of STUB1 (1:1000, 68407-1-Ig, Proteintech), PGC-1α (1:1000, 66369-1-Ig, Proteintech), TFEB (1:1000, 13372-1-AP, Proteintech), PINK1 (1:1000, ab300623, Abcam), PARKIN (1:1000, 14060-1-AP, Proteintech), LAMP-1 (1:1000, 21997-1-AP, Proteintech), Cathepsin D (1:1000, 21327-1-AP, Proteintech), Caspase3 (1:1000, 19677-1-AP, Proteintech), ubiquitin (1:1000, 10201-2-AP, Proteintech), and GAPDH (1:5000, 60004-1-Ig, Proteintech) was conducted at 4 °C overnight.

Techniques: RNA Sequencing, Ubiquitin Proteomics, Quantitative RT-PCR, Control, Immunofluorescence

Journal: European Journal of Medical Research

Article Title: STUB1-mediated PGC-1α ubiquitination promotes postoperative cognitive dysfunction in aged mice via modulating autophagy–lysosome machinery

doi: 10.1186/s40001-025-03771-1

Figure Lengend Snippet: The STUB1/PGC-1α/TFEB axis governs autophagy–lysosome machinery in the hippocampus of aged mice following anesthesia and surgery. A – I Western blot of the expression of STUB1, PGC-1α, TFEB, PINK1, PARKIN, LAMP-1, Cathepsin D, Caspase3 in the hippocampus of POCD and control mice. J , K Multiple immunofluorescence of LAMP-1 and LC3 in the hippocampus of POCD and control mice. Scale bar, 50 μm. L Transmission electron microscope of the hippocampus in POCD and control mice. Scale bar, 500 nm; n = 6 per group. *** p < 0.001; **** p < 0.0001 (Student’s t test)

Article Snippet: The membrane was rinsed by TBST (3 times × 5 min), and then slowly shaken with 5% BSA at 37 °C for 2 h. Incubation with primary antibodies of STUB1 (1:1000, 68407-1-Ig, Proteintech), PGC-1α (1:1000, 66369-1-Ig, Proteintech), TFEB (1:1000, 13372-1-AP, Proteintech), PINK1 (1:1000, ab300623, Abcam), PARKIN (1:1000, 14060-1-AP, Proteintech), LAMP-1 (1:1000, 21997-1-AP, Proteintech), Cathepsin D (1:1000, 21327-1-AP, Proteintech), Caspase3 (1:1000, 19677-1-AP, Proteintech), ubiquitin (1:1000, 10201-2-AP, Proteintech), and GAPDH (1:5000, 60004-1-Ig, Proteintech) was conducted at 4 °C overnight.

Techniques: Western Blot, Expressing, Control, Immunofluorescence, Transmission Assay, Microscopy

Journal: European Journal of Medical Research

Article Title: STUB1-mediated PGC-1α ubiquitination promotes postoperative cognitive dysfunction in aged mice via modulating autophagy–lysosome machinery

doi: 10.1186/s40001-025-03771-1

Figure Lengend Snippet: STUB1 overexpression declines the expression of PGC-1α and TFEB in mouse hippocampal neuron cells. A qRT-PCR of Stub1 transcript levels in mouse hippocampal neuron cells (HT22) transfected with Stub1 siRNA ( si-Stub1 ). B – D qRT-PCR of Stub1 , Pgc-1α , and Tfeb transcript levels in HT22 cells transfected with si- Stub1 or Stub1 overexpression ( OE-Stub1 ) plasmid. E , F Multiple-immunofluorescence of STUB1 and PGC-1α in HT22 cells transfected with si- Stub1 or OE-Stub1 plasmid. G , H Multiple-immunofluorescence of PGC-1α and TFEB in HT22 cells transfected with si- Stub1 or OE-Stub1 plasmid. Scale bar, 50 μm; n = 6 per group. ** p < 0.01; *** p < 0.001; **** p < 0.0001 (Student's t-test)

Article Snippet: The membrane was rinsed by TBST (3 times × 5 min), and then slowly shaken with 5% BSA at 37 °C for 2 h. Incubation with primary antibodies of STUB1 (1:1000, 68407-1-Ig, Proteintech), PGC-1α (1:1000, 66369-1-Ig, Proteintech), TFEB (1:1000, 13372-1-AP, Proteintech), PINK1 (1:1000, ab300623, Abcam), PARKIN (1:1000, 14060-1-AP, Proteintech), LAMP-1 (1:1000, 21997-1-AP, Proteintech), Cathepsin D (1:1000, 21327-1-AP, Proteintech), Caspase3 (1:1000, 19677-1-AP, Proteintech), ubiquitin (1:1000, 10201-2-AP, Proteintech), and GAPDH (1:5000, 60004-1-Ig, Proteintech) was conducted at 4 °C overnight.

Techniques: Over Expression, Expressing, Quantitative RT-PCR, Transfection, Plasmid Preparation, Immunofluorescence

Journal: European Journal of Medical Research

Article Title: STUB1-mediated PGC-1α ubiquitination promotes postoperative cognitive dysfunction in aged mice via modulating autophagy–lysosome machinery

doi: 10.1186/s40001-025-03771-1

Figure Lengend Snippet: STUB1 overexpression facilitates apoptosis and autophagy-lysosome machinery in mouse hippocampal neuron cells. A , B Flow cytometry for measuring apoptosis in mouse hippocampal neuron cells (HT22) transfected with Stub1 siRNA ( si-Stub1 ) or Stub1 overexpression ( OE-Stub1 ) plasmid. C , D Multiple-immunofluorescence of LAMP-1 and LC3 in HT22 cells transfected with si- Stub1 or OE-Stub1 plasmid. Scale bar, 50 μm; n = 6 per group. E Transmission electron microscope of HT22 cells transfected with si- Stub1 or OE-Stub1 plasmid. Scale bar, 1 μm. ns, p > 0.05; **** p < 0.0001 (Student’s t test)

Article Snippet: The membrane was rinsed by TBST (3 times × 5 min), and then slowly shaken with 5% BSA at 37 °C for 2 h. Incubation with primary antibodies of STUB1 (1:1000, 68407-1-Ig, Proteintech), PGC-1α (1:1000, 66369-1-Ig, Proteintech), TFEB (1:1000, 13372-1-AP, Proteintech), PINK1 (1:1000, ab300623, Abcam), PARKIN (1:1000, 14060-1-AP, Proteintech), LAMP-1 (1:1000, 21997-1-AP, Proteintech), Cathepsin D (1:1000, 21327-1-AP, Proteintech), Caspase3 (1:1000, 19677-1-AP, Proteintech), ubiquitin (1:1000, 10201-2-AP, Proteintech), and GAPDH (1:5000, 60004-1-Ig, Proteintech) was conducted at 4 °C overnight.

Techniques: Over Expression, Flow Cytometry, Transfection, Plasmid Preparation, Immunofluorescence, Transmission Assay, Microscopy

Journal: European Journal of Medical Research

Article Title: STUB1-mediated PGC-1α ubiquitination promotes postoperative cognitive dysfunction in aged mice via modulating autophagy–lysosome machinery

doi: 10.1186/s40001-025-03771-1

Figure Lengend Snippet: STUB1 overexpression reinforces PGC-1α ubiquitination to modulate autophagy-lysosome machinery. A – E qRT-PCR of Pink1 , Parkin , Lamp-1 , and Cathepsin D , and Caspase3 transcript levels in HT22 cells transfected with Stub1 siRNA ( si-Stub1 ) or Stub1 overexpression ( OE-Stub1 ) plasmid. F – N Western blot of the expression of STUB1, PGC-1α, TFEB, PINK1, PARKIN, LAMP-1, Cathepsin D, Caspase3 in HT22 cells transfected with si- Stub1 or OE-Stub1 plasmid. O Co-IP of PGC-1α ubiquitination levels in the hippocampus of POCD and control mice. P Co-IP of PGC-1α ubiquitination levels in HT22 cells transfected with si- Stub1 or OE-Stub1 plasmid. ns, p > 0.05; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001 (Student's t-test)

Article Snippet: The membrane was rinsed by TBST (3 times × 5 min), and then slowly shaken with 5% BSA at 37 °C for 2 h. Incubation with primary antibodies of STUB1 (1:1000, 68407-1-Ig, Proteintech), PGC-1α (1:1000, 66369-1-Ig, Proteintech), TFEB (1:1000, 13372-1-AP, Proteintech), PINK1 (1:1000, ab300623, Abcam), PARKIN (1:1000, 14060-1-AP, Proteintech), LAMP-1 (1:1000, 21997-1-AP, Proteintech), Cathepsin D (1:1000, 21327-1-AP, Proteintech), Caspase3 (1:1000, 19677-1-AP, Proteintech), ubiquitin (1:1000, 10201-2-AP, Proteintech), and GAPDH (1:5000, 60004-1-Ig, Proteintech) was conducted at 4 °C overnight.

Techniques: Over Expression, Ubiquitin Proteomics, Quantitative RT-PCR, Transfection, Plasmid Preparation, Western Blot, Expressing, Co-Immunoprecipitation Assay, Control