Journal: Nature Communications

Article Title: Denaturing mass photometry for rapid optimization of chemical protein-protein cross-linking reactions

doi: 10.1038/s41467-024-47732-4

Figure Lengend Snippet: Mass distributions, represented as probability density, show the evolution of monomer abundances, with measurement replicates ( n = 3) shown as overlapping curves in shades of gray. MP profiles have been measured after 5 min, 2 h, and 16 h denaturation for: ADH a in 5.4 M urea or b in 6 M guanidine HCl; GLDH c in 5.4 M urea or d in 6 M guanidine HCl and 20S proteasome e in 5.4 M urea or f in 6 M guanidine HCl. Scatter plots represent the monomer abundance (mean ± SD) after ADH, GLDH and 20S denaturation with g urea and h guanidine. Standard deviations come from measurements replicate ( n = 3). Source data are provided as a Source Data file.

Article Snippet: Human 20 S proteasome (20 S, South Bay Bio, San Jose, USA) was diluted to 1 mg/mL in 50 mM HEPES, 100 mM NaCl, pH 7.4 prior to cross-linking (see Supplementary Table for composition and molecular weights of different assemblies).

Techniques:

Journal: Nature Communications

Article Title: Denaturing mass photometry for rapid optimization of chemical protein-protein cross-linking reactions

doi: 10.1038/s41467-024-47732-4

Figure Lengend Snippet: Comparison of dMP and SDS-PAGE experiments after a ADH, c GLDH, e 20S proteasome cross-linking. Mass distributions are represented as probability density with overlapping curves in lighter shades, showing the measurement replicates ( n = 3). Relative abundances of different oligomeric states of the corresponding complex are shown as scatter plots (mean ± SD) for b ADH, d GLDH, f 20S proteasome, with error bars representing the standard deviation related to measurement replicates ( n = 3). Source data are provided as a Source Data file.

Article Snippet: Human 20 S proteasome (20 S, South Bay Bio, San Jose, USA) was diluted to 1 mg/mL in 50 mM HEPES, 100 mM NaCl, pH 7.4 prior to cross-linking (see Supplementary Table for composition and molecular weights of different assemblies).

Techniques: Comparison, SDS Page, Standard Deviation

Journal: Nature Communications

Article Title: Denaturing mass photometry for rapid optimization of chemical protein-protein cross-linking reactions

doi: 10.1038/s41467-024-47732-4

Figure Lengend Snippet: a Effect of cross-linking reagent (size, flexibility) on oligomeric states stabilized, measured in dMP: presented results are probability densities (KD) of GLDH samples cross-linked with increasing molar ratios of PhoX, DSAU, DSBU, from measurement replicates ( n = 3). b dMP-based quantitative results of XL condition screening for ADH, GLDH, and 20S proteasome complexes. Bar charts represent the dMP-calculated global inter-XL efficiency (mean ± SD) for each complex and XL condition (25/100/400:1 cross-linker:complex molar ratio), from measurement replicates ( n = 3). Plain dots represent the complex stabilization factor for each complex and XL condition. The black dash line corresponds to the stabilization factor value of 1 indicating a complex abundance similar to the native sample. Source data are provided as a Source Data file.

Article Snippet: Human 20 S proteasome (20 S, South Bay Bio, San Jose, USA) was diluted to 1 mg/mL in 50 mM HEPES, 100 mM NaCl, pH 7.4 prior to cross-linking (see Supplementary Table for composition and molecular weights of different assemblies).

Techniques:

Journal: Cell Death & Disease

Article Title: NF-kappa B interacting long noncoding RNA enhances the Warburg effect and angiogenesis and is associated with decreased survival of patients with gliomas

doi: 10.1038/s41419-020-2520-2

Figure Lengend Snippet: a LN229 and T98G cells were treated with 20(S)-Rg3 (100 μmol/L for 3 days, 100 μmol/L for 7 days, and 150 μmol/L for 3 days). Then, the expression level of NKILA was assessed using qRT-PCR. b The tube-forming capacity of LN229 and T98G cells after treatment with 20(S)-Rg3 (150 μmol/L for 3 days) was determined using HUVEC tube formation assays. c Neovascularization in LN229 and T98G cells treated with 20(S)-Rg3 (150 μmol/L for 3 days) was determined using chicken CAM assays. d Angiogenesis in vivo in LN229 cells treated with 20(S)-Rg3 was determined using the subcutaneous xenograft model and HIF-1α-, VEGFA-, and CD31-immunohistochemical staining of subcutaneous xenograft tumor tissue. e Representative 18 FDG microPET/CT images of subcutaneous xenograft tumors (LN229 cells, five mice per group) in nude mice treated with 30 mg/kg 20(S)-Rg3 for 15 consecutive days (green arrows) or phosphate-buffered saline (PBS; red arrows). Glucose uptake (SUV-bw MAX) in the subcutaneous xenograft model (five mice per group) is shown. f The expression levels of glucose GLUT1, HK2, PKM2, LDH, and MCT1 in tumors after treatment with 20(S)-Rg3 (30 mg/kg for 15 consecutive days) or PBS were determined by immunohistochemical staining. g – j Subcutaneous tumor growth rates (five mice per group) in LN229 cells with different levels of NKILA expression (LN229-O.E.-NKILA, LN229-control, LN229-20(S)-Rg3, and LN229-K.D.-NKILA). The tumor growth curves are summarized in a line chart. Average tumor weights in the subcutaneous xenograft model are shown. h Relative expression of NKILA in tumors after different treatment in vivo was assessed using qRT-PCR (n = 4). k The survival of subcutaneous tumor-bearing mice from four different groups (LN229-O.E.-NKILA, LN229-control, LN229-20(S)-Rg3, and LN229-K.D.-NKILA; n = 7) is shown in a Kaplan–Meier plot. Data are expressed as means ± SD of triplicate experiments, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: 20(S)-ginsenoside-Rg3 (C42H72O13, 785.01 g/mol) was purchased from Tauto Biotech (Shanghai, China).

Techniques: Expressing, Quantitative RT-PCR, In Vivo, Immunohistochemical staining, Staining

Journal: Pathogens

Article Title: Cell Fractionation and the Identification of Host Proteins Involved in Plant–Virus Interactions

doi: 10.3390/pathogens13010053

Figure Lengend Snippet: Representative host proteins important in plant–virus interactions that were identified via cellular fractionation.

Article Snippet: Pea or lettuce plants , LMV , 20 s Proteasome , HCPro , 30% sucrose cushion and gel filtration column , [ ] .

Techniques: Fractionation, Virus, Transgenic Assay, Membrane, Sedimentation, Centrifugation, Filtration, Fluorescence

Journal:

Article Title: Affinity Purification Strategy to Capture Human Endogenous Proteasome Complexes Diversity and to Identify Proteasome-interacting Proteins * S⃞

doi: 10.1074/mcp.M800193-MCP200

Figure Lengend Snippet: Proteins specifically interacting with the MCP21 antibody: proteasome subunits and proteasome activators/inhibitors/stabilizers Proteasome subunits listed in the table were purified as described under “Experimental Procedures” with or without formaldehyde cross-linking, separated by SDS-PAGE, and identified by nano-LC-ESI-LTQ-Orbitrap MS/MS analysis and database searching in the Swiss-Prot TrEMBL database. Nonspecific interactions could be eliminated by performing a differential analysis with proteins purified with the OX8 antibody and similarly identified. Criteria for acceptance of protein identification are described under “Experimental Procedures.” The best results in terms of protein recovery are reported. N, “normal,” i.e . without formaldehyde cross-linking; F, “formaldehyde,” i.e. with formaldehyde cross-linking.

Article Snippet: Commercially available 20 S proteasome and 26 S proteasome purified from human erythrocytes (BIOMOL International LP) were used as positive controls.

Techniques: Purification, Sequencing

Journal:

Article Title: Affinity Purification Strategy to Capture Human Endogenous Proteasome Complexes Diversity and to Identify Proteasome-interacting Proteins * S⃞

doi: 10.1074/mcp.M800193-MCP200

Figure Lengend Snippet: Differential analysis strategy to identify specific human PIPs. Erythrocytes submitted or not to in vivo cross-linking were lysed, and proteins were purified by overnight incubation at 4 °C with either the MCP21-Sepharose antibody or the OX8-Sepharose antibody as negative control. After extensive washing with 20 mm Tris-HCl, 150 mm NaCl, 1 mm EDTA, 10% glycerol, 5 mm MgCl2, 2 mm ATP, pH 7.6, proteins were eluted with a saline step using the same buffer containing 3 m NaCl. Two-milliliter fractions were collected and stored at 4 °C. Protein concentration was determined using the Bio-Rad Protein Assay, and proteasome content could be estimated by measuring the in vitro chymotrypsin-like activity as described under “Experimental Procedures.” The fractions containing the most proteasome activity were then further analyzed by Western blotting and by SDS-PAGE followed by trypsin digestion and LC-MS/MS analysis. Proteins identified in each case (experiment and negative control) were subjected to a differential analysis using the MFPaQ software (38) so that a list of specific MCP21-interacting proteins could be generated. ChT-L, ChT-like.

Article Snippet: Commercially available 20 S proteasome and 26 S proteasome purified from human erythrocytes (BIOMOL International LP) were used as positive controls.

Techniques: In Vivo, Purification, Incubation, Negative Control, Protein Concentration, In Vitro, Activity Assay, Western Blot, SDS Page, Liquid Chromatography with Mass Spectroscopy, Software, Generated

Journal:

Article Title: Affinity Purification Strategy to Capture Human Endogenous Proteasome Complexes Diversity and to Identify Proteasome-interacting Proteins * S⃞

doi: 10.1074/mcp.M800193-MCP200

Figure Lengend Snippet: Proteasome purification from human erythrocytes without (A) or with (B) in vivo formaldehyde cross-linking. Proteasome complexes were purified from human erythrocytes by affinity chromatography with the mouse IgG1 monoclonal antibody MCP21 coupled to Sepharose beads. A control purification was performed using the mouse IgG1 monoclonal antibody OX8 directed against rat CD8α. Proteins were cross-linked in vivo by incubation of human erythrocytes with 1% formaldehyde as indicated under “Experimental Procedures.” After incubation of 50 ml of erythrocyte lysate with either the MCP21-Sepharose or the OX8-Sepharose, proteins interacting with the beads were eluted by a saline step of 3 m NaCl. The eluted fractions were analyzed for their protein concentrations as well as for their proteasome contents. Statistical results were obtained from three independent experiments for each condition (formaldehyde-treated or not). Error bars indicate standard deviations (n = 3). ♦ and ⋄ represent protein concentrations in the fractions from the MCP21-Sepharose beads and from the OX8-Sepharose beads, respectively. ▴ and ▵ represent the in vitro ChT-like activity without and with 10 μm lactacystin, respectively, in the fractions eluted from the MCP21-Sepharose beads. No ChT-like activity could be detected in the fractions from the OX8 negative control experiment.

Article Snippet: Commercially available 20 S proteasome and 26 S proteasome purified from human erythrocytes (BIOMOL International LP) were used as positive controls.

Techniques: Purification, In Vivo, Affinity Chromatography, Incubation, In Vitro, Activity Assay, Negative Control

Journal:

Article Title: Affinity Purification Strategy to Capture Human Endogenous Proteasome Complexes Diversity and to Identify Proteasome-interacting Proteins * S⃞

doi: 10.1074/mcp.M800193-MCP200

Figure Lengend Snippet: Detection of 20 S core particle and 19 S activators in the purified proteasome preparations. Proteins separated by SDS-PAGE were transferred to a nitrocellulose membrane: 2 μg of commercial 20 S and 26 S proteasome from human erythrocytes as standards (A), 20 μg of total proteins from fractions containing the maximal ChT-like activity (fractions 9 and 10 from purifications without and with formaldehyde cross-linking, respectively) (B), and 10 μg of estimated 20 S proteasome (based on the ChT-like activity measurement) from fractions 8, 9, and 10 from the purification without formaldehyde cross-linking (C). Mouse monoclonal primary antibodies against 19 S proteasome subunits Rpt1 and Rpn12 and rabbit polyclonal antibodies against 20 S core subunits were used for the immunoblot staining. ECL Plex CyDye-conjugated antibodies, goat α-mouse IgG-Cy3 and goat α-rabbit IgG-Cy5, were used as secondary antibodies. The detection was performed using the Typhoon Trio fluorescence scanner at 532 nm excitation and 580 nm emission for the Cy3-conjugated antibody and 633 nm excitation and 670 nm emission for the Cy5-conjugated antibody. Lane M, molecular mass markers.

Article Snippet: Commercially available 20 S proteasome and 26 S proteasome purified from human erythrocytes (BIOMOL International LP) were used as positive controls.

Techniques: Purification, SDS Page, Activity Assay, Western Blot, Staining, Fluorescence

Journal:

Article Title: Affinity Purification Strategy to Capture Human Endogenous Proteasome Complexes Diversity and to Identify Proteasome-interacting Proteins * S⃞

doi: 10.1074/mcp.M800193-MCP200

Figure Lengend Snippet: Separation of proteasomes and proteasome-interacting proteins by SDS-PAGE. Proteins were separated by SDS-PAGE on a 8 × 10-cm acrylamide gel (12%) and detected by Coomassie Brilliant Blue staining. Whole gel lanes were analyzed. In-gel tryptic digestion of proteins was performed prior to identification by nano-LC-ESI-LTQ-Orbitrap MS/MS analysis and database searching as described under “Experimental Procedures.” Lanes 1 and 2, 2 μg of commercially available human erythrocyte 26 S and 20 S proteasomes, respectively; lanes 3 and 4, the whole protein content of eluted proteins from the OX8-Sepharose was loaded after concentration by ultrafiltration (lane 3, non formaldehyde-treated erythrocytes; lane 4, formaldehyde-treated erythrocytes); lanes 5 and 6, 40 μg of proteins from fractions 6–9 and 7–10 from purifications without and with formaldehyde cross-linking, respectively.

Article Snippet: Commercially available 20 S proteasome and 26 S proteasome purified from human erythrocytes (BIOMOL International LP) were used as positive controls.

Techniques: SDS Page, Acrylamide Gel Assay, Staining, Tandem Mass Spectroscopy, Concentration Assay

Journal:

Article Title: Affinity Purification Strategy to Capture Human Endogenous Proteasome Complexes Diversity and to Identify Proteasome-interacting Proteins * S⃞

doi: 10.1074/mcp.M800193-MCP200

Figure Lengend Snippet: Detection of 20 S core particle in the immunoprecipitated HAUSP complexes. HAUSP complexes were immunoprecipitated using the anti-HAUSP antibody or a control antibody as described under “Experimental Procedures,” separated by SDS-PAGE, and transferred to a nitrocellulose membrane. Rabbit polyclonal antibodies against 20 S core subunits were used for the immunoblot staining. Lane 1, 0.05 μg of commercially available human erythrocyte 20 S proteasome; lanes 2 and 4, whole protein content of eluted proteins from a 1.5-ml erythrocyte aliquot precipitated with the anti-HAUSP antibody (lane 2) or with the OX8 antibody (control) (lane 4); lane 3, protein sample eluted from the anti-HAUSP-Dynabeads after incubation with 1.5 ml of lysis buffer.

Article Snippet: Commercially available 20 S proteasome and 26 S proteasome purified from human erythrocytes (BIOMOL International LP) were used as positive controls.

Techniques: Immunoprecipitation, SDS Page, Western Blot, Staining, Incubation, Lysis