Journal: PLoS Pathogens

Article Title: Lu/BCAM Adhesion Glycoprotein Is a Receptor for Escherichia coli Cytotoxic Necrotizing Factor 1 (CNF1)

doi: 10.1371/journal.ppat.1003884

Figure Lengend Snippet: GST-CNF1 was incubated with buffer or with recombinant BCAM in a molar ratio CNF1∶rBCAM of 1∶1, 1∶10 and 1∶100, respectively for 20 min. The mixture was added to HeLa cells. Following 2 h incubation the cells were lysed and the CNF1-catalysed deamidation of RhoA was analyzed by the shift of the modified GTPase in SDS-PAGE by Western-blotting (A). HeLa cells were incubated with an anti Lu/BCAM antibody (AB B12) that binds to the extracellular domain or as control with an anti-Lu/BCAM antibody (AB C16) directed against the intracellular part of the glycoprotein. GST-CNF1 was then added to the cells for 2 h. We followed the toxins uptake by the amount of modified RhoA (shift in SDS-PAGE, B). Shown is a typical result of 3 independent experiments. Colocalization of DyLight488-labeled GST-CNF1 with Lu/BCAM and EEA1 (C) Top: HeLa-cells were treated on ice with DyLight488-labeled GST-CNF1 (5 mg/ml) (green) for 30 min to allow receptor binding. After 30 min cells were transferred to 37° for 30 min to induce uptake. Subsequently cells were fixed and stained for EEA1 (red) by immunofluorescence. Images are from the middle of the confocal stack. The image on the right is a maximal projection of all confocal planes. Scale bar indicates 10 µm. Middle: HeLa-cells were treated with labeled GST-CNF1 as in A. After fixation cells were stained for Lu/BCAM (red) by immunofluorescence. Images are from the middle of the confocal stack. The image on the right is a magnification of the white box. Scale bar indicates 10 µm. Bottom: HeLa-cells were treated as in A, but instead of GST-CNF1, cells were treated with DyLight488-labeled GST-CNFY (5 mg/ml) (green). After fixation cells were stained for Lu/BCAM (red) by immunofluorescence. Images are from the middle of the confocal stack. The image on the right is a magnification of the white box. Scale bar indicates 10 µm. Shown is a typical staining of 9 HeLa cells analyzed.

Article Snippet: For these studies we used recombinant Lu/BCAM, which contains a C-terminal human IgG domain for purification (Sino biology).

Techniques: Incubation, Recombinant, Modification, SDS Page, Western Blot, Labeling, Binding Assay, Staining, Immunofluorescence

Journal: PLoS Pathogens

Article Title: Lu/BCAM Adhesion Glycoprotein Is a Receptor for Escherichia coli Cytotoxic Necrotizing Factor 1 (CNF1)

doi: 10.1371/journal.ppat.1003884

Figure Lengend Snippet: A) K562 leukemia cells, which do not express Lu/BCAM (top) and the isogenic cell line K562-Lu/BCAM expressing the receptor (bottom), were treated with GST-CNF1 for different time periods from 1 h to overnight (ON) as indicated. Uptake of the toxin was analyzed by the shift of modified RhoA in SDS-PAGE. B) GST-CNF1 was incubated with buffer or with recombinant BCAM in a molar ratio CNF1∶rBCAM of 1∶1, 1∶10 and 1∶100, respectively for 20 min. The mixture was added to K562-Lu/BCAM cells for 2 h. Cells were lysed and the deamidation of RhoA was analyzed by the shift of the modified GTPase in SDS-PAGE by Western-blotting. Data are representative for at least 3 independent experiments.

Article Snippet: For these studies we used recombinant Lu/BCAM, which contains a C-terminal human IgG domain for purification (Sino biology).

Techniques: Expressing, Modification, SDS Page, Incubation, Recombinant, Western Blot

Journal: PLoS Pathogens

Article Title: Lu/BCAM Adhesion Glycoprotein Is a Receptor for Escherichia coli Cytotoxic Necrotizing Factor 1 (CNF1)

doi: 10.1371/journal.ppat.1003884

Figure Lengend Snippet: A) For dot-blots 5 µl of 3 µM solutions of GST-CNFs, GST-CNF fragments and GST alone, respectively, were spotted onto a nitrocellulose membrane. The membrane was blocked with skimmed milk and recombinant BCAM (6 µM) was added for 1 h at room temperature. Following washing bound rBCAM was detected with an anti-Lu/BCAM antibody. Equal protein load was analyzed by visualizing the GST part of the spotted proteins with an anti GST-antibody. B) Biacore protein-protein interaction studies: An antibody against human IgG (Millipore) was coupled to two lanes of a CM5-biacore chip. As ligand recombinant BCAM containing a C-terminal human IgG domain (Sino biologics) was exclusively guided over lane 2. In a second step, GST-CNF proteins as analyte were guided over both lanes. Bound protein is given as relative units (RU) corrected for the unspecific binding to lane 1 as average plus standard deviation of three independent experiments.

Article Snippet: For these studies we used recombinant Lu/BCAM, which contains a C-terminal human IgG domain for purification (Sino biology).

Techniques: Recombinant, Binding Assay, Standard Deviation

Journal: Scientific Reports

Article Title: Statins attenuate PD-L1 sorting to small extracellular vesicles dependent on ubiquitin-like 3 modification

doi: 10.1038/s41598-025-27789-x

Figure Lengend Snippet: PD-L1 is modified by UBL3. ( a ) Co-immunoprecipitation (IP) analysis of using MDA-MB-231 cell lysates expressing Flag-tagged PD-L1 (PD-L1-Flag) and biotinylated-tagged UBL3 (Biotin-UBL3). ( b ) Co-IP was performed using cell lysates expressing PD-L1-Flag or PD-L1 mutants-Flag (C250A, C272A, C250/272A) along with Biotin-UBL3 in MDA-MB-231 cells. IB analysis was conducted using the indicated antibodies. ( c ) the relative intensity of UBL3 modification (Flag/SA-HRP) calculated as the ratio of UBL3-modified PD-L1 signals (> 70 kDa) to input SA-HRP signals, normalized to the mean of cells transfected with Biotin-UBL3 and wild-type PD-L1. Data are presented as mean ± s.e.m., with dots representing individual experiments. One-way ANOVA with Tukey’s multiple comparisons test. ns, not significant, * P < 0.05, ** P < 0.001, *** P < 0.0005, PD-L1 vs. C250A, P = 0.1708; PD-L1 vs. C272A, P = 0.0009; PD-L1 vs. C250/272A, P = 0.0002; C250A vs. C272A, P = 0.0135; C272A vs. C250/272A, P = 0.5847; TMD, transmembrane domain; Flag, Flag-tag; βME-, without 2-mercaptoethanol; βME+, with 2-mercaptoethanol; SA-HRP, streptavidin-horseradish peroxidase.

Article Snippet: The antibodies used in the study were as follows: UBL3 (1:100; lab-generated; 1:1000; 14100-1-AP [Lot#00005139], Proteintech), Flag (1:1000; F3165, Sigma), streptavidin-HRP (1:5000; 19534-050, Invitrogen), GAPDH (1:1000; 2118, Cell Signaling), CD63 (1:1000; Ts63, Thermo), CD9 (1:1000; Ts9, Thermo), PD-L1 (1:500; 405.9A11, Cell Signaling), GFP (1:1000; 598, MBL) and horseradish peroxidase-conjugated IgGs (1:1000; 7076 and 7074, HRP-linked Antibody, Cell Signaling; 1:1000; 18–8816-31 and 18–8817-33, TrueBlot, Rockland).

Techniques: Modification, Immunoprecipitation, Expressing, Co-Immunoprecipitation Assay, Transfection, FLAG-tag

Journal: Scientific Reports

Article Title: Statins attenuate PD-L1 sorting to small extracellular vesicles dependent on ubiquitin-like 3 modification

doi: 10.1038/s41598-025-27789-x

Figure Lengend Snippet: UBL3 regulates PD-L1 levels in sEVs. ( a ) Immunoblot (IB) analysis. The cell lysates and sEVs from the conditioned medium of H1299 cells transfected with 3xFlag-UBL3 vectors were blotted with various antibodies. ( b ) IB analysis. The cell lysates and sEVs from the stable UBL3 knockdown H1299 cell lines were blotted with various antibodies. CD9 and CD63 were used as sEV markers, as they are tetraspanin proteins abundantly expressed on the sEV surface . Relative intensity of PD-L1 in sEVs was calculated as PD-L1/CD63 and presented as fold change, normalized to the mean value of the respective control (mock for a; shNega for b). Data are presented as mean ± s.e.m., with dots representing individual experiments. Two-tailed unpaired t-test. *** P < 0.005.

Article Snippet: The antibodies used in the study were as follows: UBL3 (1:100; lab-generated; 1:1000; 14100-1-AP [Lot#00005139], Proteintech), Flag (1:1000; F3165, Sigma), streptavidin-HRP (1:5000; 19534-050, Invitrogen), GAPDH (1:1000; 2118, Cell Signaling), CD63 (1:1000; Ts63, Thermo), CD9 (1:1000; Ts9, Thermo), PD-L1 (1:500; 405.9A11, Cell Signaling), GFP (1:1000; 598, MBL) and horseradish peroxidase-conjugated IgGs (1:1000; 7076 and 7074, HRP-linked Antibody, Cell Signaling; 1:1000; 18–8816-31 and 18–8817-33, TrueBlot, Rockland).

Techniques: Western Blot, Transfection, Knockdown, Control, Two Tailed Test

Journal: Scientific Reports

Article Title: Statins attenuate PD-L1 sorting to small extracellular vesicles dependent on ubiquitin-like 3 modification

doi: 10.1038/s41598-025-27789-x

Figure Lengend Snippet: Inhibition of UBL3 modification and PD-L1 sorting by statins. ( a and b ), Effect of pitavastatin treatment on UBL3 modification in WM9 ( a ) and H1299 ( b ) cells. Five hours after transfection, 10 µM pitavastatin was added. ( c and d ), IB analysis of the cell lysates and sEVs from the conditioned medium of H1299 cells transfected with 3xFlag-UBL3 vectors ( c ) or untransfected ( d ) were blotted with various antibodies. Five hours after gene transfection, 0.2 µM pitavastatin was added. Pit, Pitavastatin. Right panels, the relative intensity of PD-L1 in sEVs (c: PD-L1/CD63 [upper] normalized to the mean of mock − untreated control, and UBL3/CD63 [lower] normalized to the mean of Flag-UBL3 − untreated control; d: PD-L1/CD63 [upper] and UBL3/CD63 [lower], both normalized to the mean of untreated control). Data are presented as mean ± s.e.m., with dots representing individual experiments. As inhibition of UBL3 modification was observed even at low concentrations of pitavastatin (Supplementary Fig. S4b), a low concentration of pitavastatin was used in sEV purification experiments. IB analysis using the indicated antibodies. Flag, Flag-tag; GAPDH, internal control; CD63 and CD9, sEVs markers; PD-L1, endogenous PD-L1; UBL3, endogenous UBL3. βME−, without 2-mercaptoethanol. βME+, with 2-mercaptoethanol. ( c ), one-way ANOVA with Tukey’s multiple comparisons test. ( d ), Two-tailed unpaired t-test. * P < 0.05, ** P < 0.01.

Article Snippet: The antibodies used in the study were as follows: UBL3 (1:100; lab-generated; 1:1000; 14100-1-AP [Lot#00005139], Proteintech), Flag (1:1000; F3165, Sigma), streptavidin-HRP (1:5000; 19534-050, Invitrogen), GAPDH (1:1000; 2118, Cell Signaling), CD63 (1:1000; Ts63, Thermo), CD9 (1:1000; Ts9, Thermo), PD-L1 (1:500; 405.9A11, Cell Signaling), GFP (1:1000; 598, MBL) and horseradish peroxidase-conjugated IgGs (1:1000; 7076 and 7074, HRP-linked Antibody, Cell Signaling; 1:1000; 18–8816-31 and 18–8817-33, TrueBlot, Rockland).

Techniques: Inhibition, Modification, Transfection, Control, Concentration Assay, Purification, FLAG-tag, Two Tailed Test

Journal: Scientific Reports

Article Title: Statins attenuate PD-L1 sorting to small extracellular vesicles dependent on ubiquitin-like 3 modification

doi: 10.1038/s41598-025-27789-x

Figure Lengend Snippet: UBL3 and PD-L1 expression levels influence survival in lung cancer patients. ( a ) Kaplan–Meier survival curves for the lung squamous cell carcinoma cohort, stratified by UBL3 and PD-L1 expression. ( b ) Survival curves for the same cohort, stratified by UBL3 and PD-1 expression. Hazard ratios and p-values from log-rank (Mantel–Cox) tests are summarized in Supplementary Figure S7a.

Article Snippet: The antibodies used in the study were as follows: UBL3 (1:100; lab-generated; 1:1000; 14100-1-AP [Lot#00005139], Proteintech), Flag (1:1000; F3165, Sigma), streptavidin-HRP (1:5000; 19534-050, Invitrogen), GAPDH (1:1000; 2118, Cell Signaling), CD63 (1:1000; Ts63, Thermo), CD9 (1:1000; Ts9, Thermo), PD-L1 (1:500; 405.9A11, Cell Signaling), GFP (1:1000; 598, MBL) and horseradish peroxidase-conjugated IgGs (1:1000; 7076 and 7074, HRP-linked Antibody, Cell Signaling; 1:1000; 18–8816-31 and 18–8817-33, TrueBlot, Rockland).

Techniques: Expressing

Journal: eLife

Article Title: A recombinant protein containing influenza viral conserved epitopes and superantigen induces broad-spectrum protection

doi: 10.7554/eLife.71725

Figure Lengend Snippet:

Article Snippet: Peptide, recombinant protein , HA of H2N2A/Canada/720/2005 , Sino Biological , Cat: 11688-V08H , .

Techniques: Recombinant, Antibody Purification, Enzyme-linked Immunospot, Software

Journal: Protein Engineering, Design and Selection

Article Title: A fully human scFv phage display library for rapid antibody fragment reformatting

doi: 10.1093/protein/gzv024

Figure Lengend Snippet: (A) Four pairs of anti-Ncad antibody fragments were analyzed by Superdex 75 size-exclusion chromatography. The 17aa-linker and the 7aa-linker worked best for the A1 and G6 clones. For the A6 clone, the 7aa-linker diabody formed a mixture of monomer and dimer. For the D6 clone, the 17aa-linker scFv formed a mixture of monomer and dimer. (B) Four random clones from the 18aa-SX-scFv library were reformatted, expressed and purified. The 5aa-linker successfully induced dimerization for all these clones.

Article Snippet: Phage library selection The 17aa-SSA-scFv library was used for the selections against the human Ncad protein (extracellular domain fused to C-terminal polyhistidine tag, Sino Biological, cat#: 11039-H08H) using previously published methods ( Marks and Bradbury, 2004 ).

Techniques: Size-exclusion Chromatography, Clone Assay, Purification

Journal: Vaccine

Article Title: The recombinant N-terminal domain of spike proteins is a potential vaccine against Middle East respiratory syndrome coronavirus (MERS-CoV) infection

doi: 10.1016/j.vaccine.2016.11.064

Figure Lengend Snippet: Description of the N-terminal domain (NTD) immunogen and vaccination schedule. (A) The location of the NTD protein on the Middle East respiratory syndrome coronavirus MERS-CoV spike (S) protein. The recombinant (r)NTD protein consists of 336 amino acid (aa) residues (18–353) of S protein. A gp67 signal peptide (SP) was added to the N terminus for expression of the rNTD protein. (B) Purified rNTD protein detected by SDS-PAGE (left) and Western blot (right). The purified rNTD protein was separated by a 10% SDS-PAGE and stained with 0.25% Coomassie brilliant blue. Anti-NTD polyclonal antibody and infrared ray-labeled secondary antibody were used for the Western blot assay. Lane 1: protein molecular weight marker; lane 2: purified rNTD protein. (C). Vaccination schedule and detection. Mice received three vaccinations consisting of 5 or 10 μg of rNTD protein combined with adjuvants at 4-week intervals. Sera were collected at the indicated times to analyze the humoral immune response. Six mice from each group were sacrificed 2 weeks after the last immunization. The spleens were harvested for enzyme-linked immunospot (ELISpot), intracellular cytokine staining (ICS), and cytometric bead array (CBA) assays. In parallel experiments, the remaining mice were challenged with MERS-CoV to detect the protective effect elicited by the rNTD protein. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Rabbit-serum-derived polyclonal antibody against nucleoprotein (cat: 100213-RP02; Sino Biological Inc., Beijing, CHN) was incubated with the sections at 1:1000 dilution; goat anti-rabbit (cat: pv-9001; ZSGB-Bio, Beijing, CHN) secondary antibody was used at 1:2000, and sections were evaluated using light microscopy.

Techniques: Recombinant, Expressing, Purification, SDS Page, Western Blot, Staining, Labeling, Molecular Weight, Marker, Enzyme-linked Immunospot

Journal: Vaccine

Article Title: The recombinant N-terminal domain of spike proteins is a potential vaccine against Middle East respiratory syndrome coronavirus (MERS-CoV) infection

doi: 10.1016/j.vaccine.2016.11.064

Figure Lengend Snippet: IHC detection of virus antigen expression in mouse tissue after challenge with MERS-CoV. Lung (A–C) and trachea (D–F) sections were assessed using rabbit polyclonal antibody to MERS-CoV nucleoprotein (NP) 3 days after the MERS-CoV challenge. The dark purple spot marked the inflammatory cell infiltration, and the brown particle marked the antigen of MERS-CoV. The MERS-CoV was located mainly in the trachea. Additionally, the lung tissue showed MERS-CoV expression in all immunized groups.

Article Snippet: Rabbit-serum-derived polyclonal antibody against nucleoprotein (cat: 100213-RP02; Sino Biological Inc., Beijing, CHN) was incubated with the sections at 1:1000 dilution; goat anti-rabbit (cat: pv-9001; ZSGB-Bio, Beijing, CHN) secondary antibody was used at 1:2000, and sections were evaluated using light microscopy.

Techniques: Expressing