Journal: bioRxiv

Article Title: Protein SUMOylation promotes cAMP-independent EPAC1 activation

doi: 10.1101/2024.01.08.574738

Figure Lengend Snippet: Levels of cellular EPAC1 PTM probed by immunoblotting using anti-EPAC1 antibody in HUVEC (A) and HEK293/EPAC1-Flag (B) in response to heat shock as a function of time. (C) Levels of cellular EPAC1 PTM in HEK293/EPAC1-EYFP cells probed by immunoblotting using anti-EPAC1 antibody, with and without heat shock (30 min) and with or without SENP1 (220 nM) treatment at 37 °C for 20 min. Similar results were obtained from at least three independent experiments.

Article Snippet: To test if EPAC1 PTM was sensitive to SUMO-specific deconjugating enzyme, heat shock treated cells were lysed with 1× cell lysis buffer without 20 mM NEM and then incubated with 220 nM recombinant Human Sentrin-specific protease 1 (SENP1) catalytic domain (R&D Systems, Catalog no. E-700) at 37 °C for 20 min before immunoblotting analysis.

Techniques: Western Blot

Journal: The EMBO Journal

Article Title: Dual roles of HSP70 chaperone HSPA1 in quality control of nascent and newly synthesized proteins

doi: 10.15252/embj.2020106183

Figure Lengend Snippet: Cell lysates were prepared from MCF7 cells maintained at 37°C or heat‐shocked at 42°C for 1 h. Where indicated, lysate from heat‐shocked cells was incubated with recombinant USP2 to remove polyubiquitin chains. Equal amounts of cell lysate were assayed for the levels of the indicated proteins by immunoblotting. A quantification of the levels of K48‐linked polyubiquitin is shown in the bar graph (means ± SD, n = 4, number indicates P value calculated with the two‐stage linear step‐up procedure of Benjamini, Krieger, and Yekutieli). Equal amounts of cell lysate described in (A) were fractionated by sucrose density gradient centrifugation, and K48‐linked polyubiquitin was detected by immunoblotting. Quantification of K48‐linked polyubiquitin across the gradients relative to the levels obtained in MCF7 cells maintained at 37°C (means ± SEM, n = 3). Trypsin‐like proteasome activity determined in the same fractions as in (B). Results are shown relative to the proteasome activity obtained in MCF7 cells maintained at 37°C (means ± SEM, n = 3). Source data are available online for this figure.

Article Snippet: To digest K48‐polyubiquitin chains, the lysate was incubated with 7 μg of recombinant human USP2 catalytic domain protein (Boston Biochem, E‐506) for 1 h at 25°C.

Techniques: Incubation, Recombinant, Western Blot, Gradient Centrifugation, Activity Assay

Journal: The EMBO Journal

Article Title: Dual roles of HSP70 chaperone HSPA1 in quality control of nascent and newly synthesized proteins

doi: 10.15252/embj.2020106183

Figure Lengend Snippet:

Article Snippet: To digest K48‐polyubiquitin chains, the lysate was incubated with 7 μg of recombinant human USP2 catalytic domain protein (Boston Biochem, E‐506) for 1 h at 25°C.

Techniques: Recombinant, Sequencing, Magnetic Beads, Staining, Protease Inhibitor, DNA Purification, Bicinchoninic Acid Protein Assay, Modification, Software, Imaging

Journal: Science Advances

Article Title: PELP1 coordinates the modular assembly and enzymatic activity of the rixosome complex

doi: 10.1126/sciadv.adw4603

Figure Lengend Snippet: ( A ) Size exclusion chromatography (SEC) curves exhibiting the formation of a complex between SENP3 protease domain 302 to 574aa and PELP1 SLiM -containing peptide 761 to 796aa. SDS-PAGE and total protein staining of SEC fractions are displayed below the x axis. ( B ) AlphaFold3 structural models of proSUMO substrates used in endopeptidase cleavage assays. Sequences of the proSUMO C-terminal tails (cleavage sites labeled with arrows) are shown below the structural models. ( C ) SDS-PAGE and total protein stain of in vitro SENP3 ± MBP-PELP1 SLiM endopeptidase activity assays against proSUMO1, −2, and −3 substrates. Decreasing concentrations (2500 to 0.12 nM) of SENP3 enzyme ± MBP-PELP1 761 to 796aa was incubated for 1 hour at 37°C with 5 μM proSUMO substrate. Cleaved proSUMO product is labeled with asterisks. Quantification curves representing percent (%) proSUMO endopeptidase cleavage correspond to the gel images and the enzyme concentration range was 1000 to 15.63 nM. SD was calculated from three independent experiments ( n = 3). ( D ) SDS-PAGE and total protein stain of in vitro SENP3 (C532A) ± MBP-PELP1 SLiM endopeptidase activity assay, illustrating no activity. ( E ) Quantification curves representing percent (%) proSUMO2 endopeptidase cleavage by SENP protease domain + PELP1 SLiM , SENP3 protease domain alone, or SENP5 protease domain alone during a time course (0 to 450 s). Enzyme concentration was kept constant at 1000 nM along with substrate concentration at 5 μM. Percent cleavage was calculated as in (C). SD was calculated from three independent assay samples ( n = 3). Raw gel images are displayed in fig. S13A. ( F ) Differential scanning fluorimetry curves exhibiting the thermal stabilization of the SENP3 protease domain upon addition of short (“s,” amino acids 764 to 781) and long (“l,” amino acids 764 to 792) PELP1 SLIM peptides. Boltzmann and first derivative curves are shown on the top and bottom of the panel, respectively. Values for change in T m is only shown for the first derivative.

Article Snippet: N-terminal 6× His-tagged human SENP3 protease domain (302 to 574aa, E. coli codon-optimized gene synthesized by Genscript in pET11a vector) and SENP5 protease domain [567 to 755aa, plasmid no. 16358 ( ) from Addgene in pET28a vector] were used for in vitro binding and deSUMOylation assays.

Techniques: Size-exclusion Chromatography, SDS Page, Staining, Labeling, In Vitro, Activity Assay, Incubation, Concentration Assay