Journal: Cell Death & Disease

Article Title: PTPN23 ubiquitination by WDR4 suppresses EGFR and c-MET degradation to define a lung cancer therapeutic target

doi: 10.1038/s41419-023-06201-4

Figure Lengend Snippet: A Schematic presentation of the design of quantitative proteome and ubiquitylome analyses on control and WDR4 overexpressing (OE) A549 cells or control and WDR4 knockdown A549 cells. The criteria for hit selection and the number of hits recovered are shown. B , Immunoprecipitation analysis for PTPN23 ubiquitination in HEK293T ( B ) and H1299 cells transfected with the indicated constructs. D , E Immunoprecipitation analysis of PTPN23 ubiquitination in H1299 cells stably expressing control or WDR4 shRNAs ( D ) or A549 control or WDR4 KO cells ( E ) and transiently transfected with the indicated constructs. The efficient knockdown and knockout of WDR4 in these cells are shown in Figs. B and , respectively. F , G Immunoprecipitation analysis of the interaction between endogenous PTPN23 and endogenous WDR4 in indicated cells. H Representative PLA images for the interaction between endogenous WDR4 and endogenous PTPN23 in H1299 cells. Antibodies used are indicated. Bar, 10 μm. I GST pull down analysis for the in vitro interaction between GST-WDR4 and His-PTPN23. J In vitro ubiquitination assay for PTPN23. His-PTPN23 purified from baculovirus was incubated with E1, E2, ubiquitin and/or WDR4-based Cul4A or Cul4B complex purified from transfected HEK293T cells. The integrity of input E3 ligase complex is shown on the right.

Article Snippet: K-ε-GG peptides were enriched by PTMScan Ubiquitin Remnant Motif Kit (Cell Signaling Technology) according to manufacturer’s instructions.

Techniques: Selection, Immunoprecipitation, Transfection, Construct, Stable Transfection, Expressing, Knock-Out, In Vitro, Ubiquitin Assay, Purification, Incubation

Journal: bioRxiv

Article Title: Proteome birthdating reveals age-selectivity of protein ubiquitination

doi: 10.1101/2023.10.08.561433

Figure Lengend Snippet: A) SDS-PAGE and anti-ubiquitin western blots showing the accumulation of ubiquitinated proteins in human fibroblasts after addition of MG132 at different concentrations for 6 hours. Each concentration point was analyzed in duplicate experiments. B) Coverage statistics of proteome birthdating experiments for Kε-GG and unmodified peptides. Details of the analyses are described in Materials and Methods. Datasets of the measured parameters are tabulated in Supplementary Table S2. C) Pairwise comparison of measured median ages of Kε-GG and unmodified peptides mapped to the same proteins. Green and purple dashed boxes highlight Kε-GG peptides that are, respectively, younger or older than their unmodified counterparts mapped to the same proteins. D) Log 2 ratios of ages of Kε-GG and unmodified peptides mapped to the same proteins.

Article Snippet: Kε-GG peptides were enriched using the PTMScan HS Ubiquitin/SUMO Remnant Motif (Kε-GG) Kit from Cell Signaling Technologies using the provided reagents and protocol.

Techniques: SDS Page, Western Blot, Concentration Assay, Comparison

Journal: bioRxiv

Article Title: Proteome birthdating reveals age-selectivity of protein ubiquitination

doi: 10.1101/2023.10.08.561433

Figure Lengend Snippet: A) Coverage statistics for proteome-wide changes in Kε-GG peptide levels upon proteasome inhibition. Datasets of the measured parameters are tabulated in Supplementary Table S3. B) Intensity distributions of Kε-GG and unmodified peptides in the presence and absence of MG132. The plots indicate that Kε-GG peptides, but not unmodified peptides, increase their levels upon proteasomal inhibition. C) Rank-size distribution plots showing Log 2 fold increases in levels of Kε-GG peptides upon proteasomal inhibition. Vertical columns of blue points on the plot represent data for peptides matched to specific proteins. The green points are median peptide-level measurements for each protein. Proteins are rank ordered based on median peptide measurements. Note that MG132-induced changes in levels of Kε-GG peptides mapped to the same proteins are highly variable. D) Log 2 fold increases in levels of Kε-GG peptides mapped to ubiquitin upon proteasomal inhibition. E) Correlation between MG132-induced changes in levels of Kε-GG peptides and their age. F) Log 2 ratios of ages of Kε-GG and unmodified peptides mapped to the same proteins. In E and F, peptides whose levels increased by greater or less than a factor of four are shown in purple and green colors, respectively. The analysis in E and F indicate that, in general, younger Kε-GG peptides accumulate more in the presence of MG132 in comparison to older Kε-GG peptides.

Article Snippet: Kε-GG peptides were enriched using the PTMScan HS Ubiquitin/SUMO Remnant Motif (Kε-GG) Kit from Cell Signaling Technologies using the provided reagents and protocol.

Techniques: Inhibition, Comparison