Journal: mAbs

Article Title: Beyond bispecificity: Controlled Fab arm exchange for the generation of antibodies with multiple specificities

doi: 10.1080/19420862.2021.2018960

Figure Lengend Snippet: Generation of multispecific DB-VHH constructs. Trastuzumab IgG1-K409R mAb (anti-HER2) C-termini are fused to VHHs directed against EGFR, IL6R or NKG2D via a GS-linker. CS06 IgG1-F405L mAb (anti-c-MET) is fused to the same VHH molecules in the same manner. After recombinant production and purification, parental IgG-VHHs are recombined pairwise by reduction and reoxidation. The matching K409R and F405L mutations drive the generation of heterodimeric multispecific DB-VHHs.

Article Snippet: The biosensors were then rinsed in kinetics buffer (KB; PBS + 0.1% Tween-20 + 1% bovine serum albumin (BSA)) for 45 s (baseline) and the corresponding antigen was associated to the biosensor for 300 s. Different antigen concentrations (1:2 dilution rows in KB) were tested in order to identify the dynamic range of the binding kinetics and the most suitable concentration ranges were determined as follows: HER2/ErbB2 ECD-His Tag (Sino Biological, Cat: 10004- H08H) – 0.78 nM to 50 nM, c-MET ECD-strepII-His Tag (in-house production) – 0.39 nM to 25 nM, human EGFR-His Tag (ACRO Biosystems, Cat: EGR-H5222) – 1.5 nM to 100 nM, human IL6R ECD-His Tag (Sino Biological, Cat: 10398-H08H) – 0.78 nM to 50 nM, and human NKG2D-His Tag (Sino Biological, Cat: 10575-H07B) – 0.78 nM to 50 nM.

Techniques: Construct, Recombinant, Purification

Journal: mAbs

Article Title: Beyond bispecificity: Controlled Fab arm exchange for the generation of antibodies with multiple specificities

doi: 10.1080/19420862.2021.2018960

Figure Lengend Snippet: Binding affinities measured for each paratope using recombinant antigens

Article Snippet: The biosensors were then rinsed in kinetics buffer (KB; PBS + 0.1% Tween-20 + 1% bovine serum albumin (BSA)) for 45 s (baseline) and the corresponding antigen was associated to the biosensor for 300 s. Different antigen concentrations (1:2 dilution rows in KB) were tested in order to identify the dynamic range of the binding kinetics and the most suitable concentration ranges were determined as follows: HER2/ErbB2 ECD-His Tag (Sino Biological, Cat: 10004- H08H) – 0.78 nM to 50 nM, c-MET ECD-strepII-His Tag (in-house production) – 0.39 nM to 25 nM, human EGFR-His Tag (ACRO Biosystems, Cat: EGR-H5222) – 1.5 nM to 100 nM, human IL6R ECD-His Tag (Sino Biological, Cat: 10398-H08H) – 0.78 nM to 50 nM, and human NKG2D-His Tag (Sino Biological, Cat: 10575-H07B) – 0.78 nM to 50 nM.

Techniques: Binding Assay, Recombinant, Construct

Journal: mAbs

Article Title: Beyond bispecificity: Controlled Fab arm exchange for the generation of antibodies with multiple specificities

doi: 10.1080/19420862.2021.2018960

Figure Lengend Snippet: Biolayer interferometry analysis of simultaneous antigen binding of tri- and tetraspecific DB-VHHs. (a), (b) and (c) show exemplary sensorgrams for trispecific molecules and (d) a tetraspecific DB-VHH. The first association step represents binding of the DB-VHH (200 nM) via its CS06 paratope to biotinylated c-MET immobilized to streptavidin biosensors. Second (and third for (D)) association step is performed using an IL6R, EGFR or NKG2D recombinant protein (200 nM). The last association step is performed using HER2 (200 nM). Kinetic buffer (KB) controls were applied as negative controls for each association step.

Article Snippet: The biosensors were then rinsed in kinetics buffer (KB; PBS + 0.1% Tween-20 + 1% bovine serum albumin (BSA)) for 45 s (baseline) and the corresponding antigen was associated to the biosensor for 300 s. Different antigen concentrations (1:2 dilution rows in KB) were tested in order to identify the dynamic range of the binding kinetics and the most suitable concentration ranges were determined as follows: HER2/ErbB2 ECD-His Tag (Sino Biological, Cat: 10004- H08H) – 0.78 nM to 50 nM, c-MET ECD-strepII-His Tag (in-house production) – 0.39 nM to 25 nM, human EGFR-His Tag (ACRO Biosystems, Cat: EGR-H5222) – 1.5 nM to 100 nM, human IL6R ECD-His Tag (Sino Biological, Cat: 10398-H08H) – 0.78 nM to 50 nM, and human NKG2D-His Tag (Sino Biological, Cat: 10575-H07B) – 0.78 nM to 50 nM.

Techniques: Binding Assay, Recombinant

Journal: mAbs

Article Title: Beyond bispecificity: Controlled Fab arm exchange for the generation of antibodies with multiple specificities

doi: 10.1080/19420862.2021.2018960

Figure Lengend Snippet: Specific cell clustering due to simultaneous binding of the DB-VHHs to three different cancer cell lines. Flow cytometry cytograms represent the fluorescence signals of the different cell populations. (a) Cells without antibody construct. Upper left gate = HCC-1954 (HER2 +++ ) cells stained with DeepRed, lower left gate = MDA-MB-468 (EGFR +++ ) cells stained with CMRA, lower right gate = EBC-1 (c-MET ++ ) cells stained with CFSE, upper right gate = HCC-1954 + EBC-1 cell doublets. Events in all three fluorescence channels (cell triplets) were marked in red. Cells were incubated in the presence of 1 nM (b) bispecific DB, (c) and (d) trispecific DB-VHHs, (e) and (f) tetraspecific DB-VHHs.

Article Snippet: The biosensors were then rinsed in kinetics buffer (KB; PBS + 0.1% Tween-20 + 1% bovine serum albumin (BSA)) for 45 s (baseline) and the corresponding antigen was associated to the biosensor for 300 s. Different antigen concentrations (1:2 dilution rows in KB) were tested in order to identify the dynamic range of the binding kinetics and the most suitable concentration ranges were determined as follows: HER2/ErbB2 ECD-His Tag (Sino Biological, Cat: 10004- H08H) – 0.78 nM to 50 nM, c-MET ECD-strepII-His Tag (in-house production) – 0.39 nM to 25 nM, human EGFR-His Tag (ACRO Biosystems, Cat: EGR-H5222) – 1.5 nM to 100 nM, human IL6R ECD-His Tag (Sino Biological, Cat: 10398-H08H) – 0.78 nM to 50 nM, and human NKG2D-His Tag (Sino Biological, Cat: 10575-H07B) – 0.78 nM to 50 nM.

Techniques: Binding Assay, Flow Cytometry, Fluorescence, Construct, Staining, Incubation

Journal: mAbs

Article Title: Beyond bispecificity: Controlled Fab arm exchange for the generation of antibodies with multiple specificities

doi: 10.1080/19420862.2021.2018960

Figure Lengend Snippet: Simultaneous interaction of the DB-VHHs with HCC-1954 and EBC-1 target cells and binding of recombinant IL6R. Flow cytometry cytograms represent the fluorescence signals of the two cell populations and bound recombinant IL6R, detected with an anti-His6 detection antibody. (a) Cells without antibody construct. Upper left gate = HCC-1954 (HER2+++) cells stained with DeepRed, lower right gate = EBC-1 (c-MET++) cells stained with CFSE, upper right gate = HCC-1954 + EBC-1 cell doublets. Events in all three fluorescence channels (HCC-1954 + EBC-1 + bound recombinant IL6R-His Tag) were marked in blue. Cells were incubated in the presence of 10 nM (b) bispecific DB, (c) and (d) trispecific DB-VHHs, (e) and (f) tetraspecific DB-VHHs.

Article Snippet: The biosensors were then rinsed in kinetics buffer (KB; PBS + 0.1% Tween-20 + 1% bovine serum albumin (BSA)) for 45 s (baseline) and the corresponding antigen was associated to the biosensor for 300 s. Different antigen concentrations (1:2 dilution rows in KB) were tested in order to identify the dynamic range of the binding kinetics and the most suitable concentration ranges were determined as follows: HER2/ErbB2 ECD-His Tag (Sino Biological, Cat: 10004- H08H) – 0.78 nM to 50 nM, c-MET ECD-strepII-His Tag (in-house production) – 0.39 nM to 25 nM, human EGFR-His Tag (ACRO Biosystems, Cat: EGR-H5222) – 1.5 nM to 100 nM, human IL6R ECD-His Tag (Sino Biological, Cat: 10398-H08H) – 0.78 nM to 50 nM, and human NKG2D-His Tag (Sino Biological, Cat: 10575-H07B) – 0.78 nM to 50 nM.

Techniques: Binding Assay, Recombinant, Flow Cytometry, Fluorescence, Construct, Staining, Incubation

Journal: mAbs

Article Title: Beyond bispecificity: Controlled Fab arm exchange for the generation of antibodies with multiple specificities

doi: 10.1080/19420862.2021.2018960

Figure Lengend Snippet: DB-VHHs elicit potent and specific NK cell-mediated target cell killing. Tumor cells were incubated with primary effector cells (NK cells) at a 1:5 ratio in the presence of antibody constructs in different concentrations. Error bars represent standard deviation of two biological replicates. Wildtype CS06 and trastuzumab with active Fc effector functioning were used as an ADCC reference (green). (a) and (b): c-MET-positive EBC-1 target cells, (c) and (d) HER2-overexpressing SK-BR-3 target cells. (a) and (c): NK cell cytotoxicity triggered by parental antibodies. (b) and (d): DB-VHHs serve as NK cell engager and mediate tumor cell killing.

Article Snippet: The biosensors were then rinsed in kinetics buffer (KB; PBS + 0.1% Tween-20 + 1% bovine serum albumin (BSA)) for 45 s (baseline) and the corresponding antigen was associated to the biosensor for 300 s. Different antigen concentrations (1:2 dilution rows in KB) were tested in order to identify the dynamic range of the binding kinetics and the most suitable concentration ranges were determined as follows: HER2/ErbB2 ECD-His Tag (Sino Biological, Cat: 10004- H08H) – 0.78 nM to 50 nM, c-MET ECD-strepII-His Tag (in-house production) – 0.39 nM to 25 nM, human EGFR-His Tag (ACRO Biosystems, Cat: EGR-H5222) – 1.5 nM to 100 nM, human IL6R ECD-His Tag (Sino Biological, Cat: 10398-H08H) – 0.78 nM to 50 nM, and human NKG2D-His Tag (Sino Biological, Cat: 10575-H07B) – 0.78 nM to 50 nM.

Techniques: Incubation, Construct, Standard Deviation

Journal: mAbs

Article Title: Beyond bispecificity: Controlled Fab arm exchange for the generation of antibodies with multiple specificities

doi: 10.1080/19420862.2021.2018960

Figure Lengend Snippet:

Article Snippet: The biosensors were then rinsed in kinetics buffer (KB; PBS + 0.1% Tween-20 + 1% bovine serum albumin (BSA)) for 45 s (baseline) and the corresponding antigen was associated to the biosensor for 300 s. Different antigen concentrations (1:2 dilution rows in KB) were tested in order to identify the dynamic range of the binding kinetics and the most suitable concentration ranges were determined as follows: HER2/ErbB2 ECD-His Tag (Sino Biological, Cat: 10004- H08H) – 0.78 nM to 50 nM, c-MET ECD-strepII-His Tag (in-house production) – 0.39 nM to 25 nM, human EGFR-His Tag (ACRO Biosystems, Cat: EGR-H5222) – 1.5 nM to 100 nM, human IL6R ECD-His Tag (Sino Biological, Cat: 10398-H08H) – 0.78 nM to 50 nM, and human NKG2D-His Tag (Sino Biological, Cat: 10575-H07B) – 0.78 nM to 50 nM.

Techniques: Variant Assay, Hydrophobic Interaction Chromatography, Mutagenesis, Molecular Weight, High Performance Liquid Chromatography

Journal: Journal of Biological Chemistry

Article Title: Phosphorylation of Targeting Protein for Xenopus Kinesin-like Protein 2 (TPX2) at Threonine 72 in Spindle Assembly

doi: 10.1074/jbc.m114.591545

Figure Lengend Snippet: FIGURE 1. The evolutionary conserved Thr72 in human TPX2 is phosphory- lated by Cdk1 and Cdk2 in vitro. A, comparative alignment of part of human TPX2 sequence (amino acids 41 to 80) with corresponding sequences from other species (mouse, rat, and frog) using DNAMAN software (Lynnon Corpo- ration). The sequence alignment shows that Thr72 in human TPX2 is con- served in all other species. B, schematic diagram of the experimental protocol for mass spectrometry analysis (LC-MS/MS) using mitotic HeLa cells. HeLa cells were synchronized at M phase by nocodazole treatment (100 ng/ml) for 16 h and released for 30 min after nocodazole washout. Endogenous TPX2 was immunoprecipitated from 10 mg of total protein using pan-TPX2 Abs (clone 184). IP sample was run on SDS-PAGE and after Coomassie Blue stain- ing, the band with the matching size to TPX2 (confirmed by Western blotting with TPX2 Abs, not shown) was cut out and sent for LC-MS/MS analysis. The gray asterisk on the spectra of the phosphopeptide containing Thr72 indicate the identified matched fragment ions on mass spectrometry. C, phosphoryl- ation sites identified by mass spectrometry analysis on endogenous TPX2 immunoprecipitated from nocodazole-synchronized mitotic HeLa cells in regards to the known TPX2 domains. All these sites have been identified previously (17, 19–32) but not confirmed and analyzed. Thr72 is the first vali- dated and functionally characterized phosphorylation site in human TPX2 (this study). D, in vitro kinase assay using purified Cdk1/2 proteins and phos- phospecific Thr72 TPX2 Abs. Purified GST fusion protein TPX2 WT, GST-TPX2- T72A, or GST-TPX2-T72E was incubated with each active Cdk-cyclin complex inthepresenceof1mMcoldATP.Allkinasereactionswerestoppedbyadding 2 SDS sample buffer and the samples were run on SDS-polyacrylamide gel electrophoresis followed by Western blot detection using the indicated antibodies.

Article Snippet: Antibodies—Primary Abs against Cdk1, Cdk2 (Santa Cruz), Thr(P)72 TPX2 (homemade, described above), Cyclin B (Abcam), 9124 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 290 • NUMBER 14 • APRIL 3, 2015 at H A R V A R D U N IV E R SIT Y on A pril 15, 2015 http://w w w .jbc.org/ D ow nloaded from -actin (Chemicon), TPX2 (clone 183, epitope: 150 –200 amino acids of human TPX2, Novus Biologicals; clone 184, epitope: 700 –749 amino acids of TPX2, Novus Biologicals), phosphoCdk/MAPK substrates (34B2, Cell Signaling), Cy3 conjugated-tubulin (Sigma), p-Aurora A (ab18318, Abcam), and monoclonal TPX2 (18D5-1, Abcam) were used for Western blots, immunoprecipitations, and/or immunofluorescence experiments.

Techniques: In Vitro, Sequencing, Software, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Immunoprecipitation, SDS Page, Staining, Western Blot, Phospho-proteomics, Kinase Assay, Purification, Incubation, Polyacrylamide Gel Electrophoresis

Journal: Journal of Biological Chemistry

Article Title: Phosphorylation of Targeting Protein for Xenopus Kinesin-like Protein 2 (TPX2) at Threonine 72 in Spindle Assembly

doi: 10.1074/jbc.m114.591545

Figure Lengend Snippet: FIGURE 2. Thr(P)72 TPX2 antibodies are specific in Western blot for TPX2 phosphorylated at Thr72 in vivo. A, specificity of Thr(P)72 TPX2 for TPX2 proteintestedbysiRNA.HeLacellsweretransfectedwithcontrolsiRNAorone of two TPX2 siRNAs for 24 h and synchronized at M phase with nocodazole treatment (100 ng/ml). Cells were harvested and lysed with lysis buffer. Sam- ples were run on SDS-PAGE, followed by Western blotting, first probed with the Thr(P)72 TPX2 Abs, then stripped and re-probed with pan-TPX2 (clone 184) Abs. Levels of actin were used as loading controls. B, bar graph quanti- tation for the relative expression levels of Thr(P)72 TPX2 and TPX2 in control and TPX2 siRNA-transfected cells. Each sample was compared with sample treated with control siRNA. Relative expression levels of Thr(P)72 for control siRNA,10;TPX2siRNA#1(UTR),0.3180.085;TPX2siRNA#2(Cds),0.289 0.115. Relative expression levels of TPX2, control siRNA, 1 0; TPX2 siRNA #1, 0.335 0.0074; TPX2 siRNA #2, 0.304 0.091 (mean S.E.). n 4 samples, from 4 independent experiments. Unpaired Student’s t test indicated all the results are significant. ***, p 0.001. C, specificity of Thr(P)72 TPX2 tested by the use of T72A mutant and -PPase treatment. HeLa cells were left untrans- fected, transfected with an empty GFP vector, GFP-TPX2 WT, or GFP-TPX2 T72A mutant plasmids. 24 h after transfection, cells were synchronized with nocodazole for 16 h, harvested, and lysed. TPX2 immunoprecipitation was performed in each sample with TPX2 Abs (clone 183). Where indicated, IP

Article Snippet: Antibodies—Primary Abs against Cdk1, Cdk2 (Santa Cruz), Thr(P)72 TPX2 (homemade, described above), Cyclin B (Abcam), 9124 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 290 • NUMBER 14 • APRIL 3, 2015 at H A R V A R D U N IV E R SIT Y on A pril 15, 2015 http://w w w .jbc.org/ D ow nloaded from -actin (Chemicon), TPX2 (clone 183, epitope: 150 –200 amino acids of human TPX2, Novus Biologicals; clone 184, epitope: 700 –749 amino acids of TPX2, Novus Biologicals), phosphoCdk/MAPK substrates (34B2, Cell Signaling), Cy3 conjugated-tubulin (Sigma), p-Aurora A (ab18318, Abcam), and monoclonal TPX2 (18D5-1, Abcam) were used for Western blots, immunoprecipitations, and/or immunofluorescence experiments.

Techniques: Western Blot, In Vivo, Lysis, SDS Page, Expressing, Control, Transfection, Mutagenesis, Plasmid Preparation, Immunoprecipitation

Journal: Journal of Biological Chemistry

Article Title: Phosphorylation of Targeting Protein for Xenopus Kinesin-like Protein 2 (TPX2) at Threonine 72 in Spindle Assembly

doi: 10.1074/jbc.m114.591545

Figure Lengend Snippet: FIGURE 3. In vivo TPX2 phosphorylation at Thr72 is cell cycle-dependent and peaks at M phase. A, cell cycle profiles analyzed by flow cytometry analysis to confirmcellsynchronizationateachphase.B,afterIPswithpan-TPX2Abs(clone184),WesternblotswereprobedfirstwiththeThr(P)72TPX2Absandthen,after stripping, re-probed with pan-TPX2 Abs (clone 184). The levels of -actin were used as loading controls. The levels of cyclin B1 were used as positive controls to show that synchronization at M phase worked well, as indicated by the high level of cyclin B1 in M phase compared with that of S phase or non-synchronized cells. The Western blot figures are representative of 3 independent experiments. The input blot is from the mitotic samples. C, bar graphs for quantification of relative levels of Thr72 phosphorylation are shown. Non-syn, non-synchronized cells (1 0); M, M phase cells (4.16 0.36); S, S phase cells (1.31 0.4); mean of the relative levels of Thr(P)72 TPX2/TPX2 expression S.D., n 3 independent experiments; ***, non-syn versus M phase, p 0.001; **, M versus S phase, p 0.01; NS, not significant: non-syn versus S phase, all by unpaired Student’s t test.

Article Snippet: Antibodies—Primary Abs against Cdk1, Cdk2 (Santa Cruz), Thr(P)72 TPX2 (homemade, described above), Cyclin B (Abcam), 9124 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 290 • NUMBER 14 • APRIL 3, 2015 at H A R V A R D U N IV E R SIT Y on A pril 15, 2015 http://w w w .jbc.org/ D ow nloaded from -actin (Chemicon), TPX2 (clone 183, epitope: 150 –200 amino acids of human TPX2, Novus Biologicals; clone 184, epitope: 700 –749 amino acids of TPX2, Novus Biologicals), phosphoCdk/MAPK substrates (34B2, Cell Signaling), Cy3 conjugated-tubulin (Sigma), p-Aurora A (ab18318, Abcam), and monoclonal TPX2 (18D5-1, Abcam) were used for Western blots, immunoprecipitations, and/or immunofluorescence experiments.

Techniques: In Vivo, Phospho-proteomics, Flow Cytometry, Stripping Membranes, Western Blot, Expressing

Journal: Journal of Biological Chemistry

Article Title: Phosphorylation of Targeting Protein for Xenopus Kinesin-like Protein 2 (TPX2) at Threonine 72 in Spindle Assembly

doi: 10.1074/jbc.m114.591545

Figure Lengend Snippet: FIGURE 4. Phosphorylation of TPX2 at Thr72 is inhibited by the Cdk inhibitor roscovitine. A, the levels of Thr(P)72 were reduced by roscovitine treatment. HeLa cells were first treated with 100 ng/ml of nocodazole for 16 h and then once synchronized, treated with dimethyl sulfoxide (as a control), or 20 and 40 M roscovitine for 30 min. Cells were harvested, lysed, and TPX2 was immunoprecipitated with pan-TPX2 Abs (clone 184). SDS-PAGE was performed and followed by Western blotting with Thr(P)72 and pan-TPX2 Abs (clone 184). B, cyclin B1 levels were also used to confirm that roscovitine-treated cells had remained in mitosis. The levels of p-Cdk/MAPK substrates (PX(S*/T*)P or (S*/T*)PX(R/K) motif) were also used to confirm the effectiveness of the treatment. Actin levels were used as loading control.

Article Snippet: Antibodies—Primary Abs against Cdk1, Cdk2 (Santa Cruz), Thr(P)72 TPX2 (homemade, described above), Cyclin B (Abcam), 9124 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 290 • NUMBER 14 • APRIL 3, 2015 at H A R V A R D U N IV E R SIT Y on A pril 15, 2015 http://w w w .jbc.org/ D ow nloaded from -actin (Chemicon), TPX2 (clone 183, epitope: 150 –200 amino acids of human TPX2, Novus Biologicals; clone 184, epitope: 700 –749 amino acids of TPX2, Novus Biologicals), phosphoCdk/MAPK substrates (34B2, Cell Signaling), Cy3 conjugated-tubulin (Sigma), p-Aurora A (ab18318, Abcam), and monoclonal TPX2 (18D5-1, Abcam) were used for Western blots, immunoprecipitations, and/or immunofluorescence experiments.

Techniques: Phospho-proteomics, Control, Immunoprecipitation, SDS Page, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Phosphorylation of Targeting Protein for Xenopus Kinesin-like Protein 2 (TPX2) at Threonine 72 in Spindle Assembly

doi: 10.1074/jbc.m114.591545

Figure Lengend Snippet: FIGURE 5. Localization of Thr(P)72 TPX2 in HeLa and 293 cells. Mitotic (A and B) and interphase (C) HeLa cells were stained with Abs directed against Thr(P)72 TPX2, TPX2, and tubulin or with the Thr(P)72 Abs pre-absorbed with blocking peptide at different ratios. A, in mitotic cells, TPX2 phosphorylated at Thr72 is localized in the cytosol and does not strictly associate with the mitotic spindle. B, HeLa cells stained with pan-TPX2 and Thr(P)72 TPX2 Abs preincubated with Thr(P)72 blocking peptides. C, during interphase, Thr(P)72 TPX2 is localized in the nucleus. Note that the expression levels of Thr(P)72 are much lower in interphase cells than in mitotic cells. Note that only the Thr(P)72 signal was blocked. D, representative photographs of mitotic 293 cells transfected with GFP-TPX2 WT (WT) and GFP-TPX2 T72A (T72A). Scatter plots show the GFP signal at microtubules relative to total GFP signal. GFP-TPX2 T72A is significantly enriched on microtubules when compared with GFP-TPX2 WT (GFP-TPX2 WT (0.26 0.01, n 29) versus GFP-TPX2 T72A (0.39 0.02, n 22), group (mean S.E.); ***, p 0.0001 by t test). Scale bar, 10 m.

Article Snippet: Antibodies—Primary Abs against Cdk1, Cdk2 (Santa Cruz), Thr(P)72 TPX2 (homemade, described above), Cyclin B (Abcam), 9124 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 290 • NUMBER 14 • APRIL 3, 2015 at H A R V A R D U N IV E R SIT Y on A pril 15, 2015 http://w w w .jbc.org/ D ow nloaded from -actin (Chemicon), TPX2 (clone 183, epitope: 150 –200 amino acids of human TPX2, Novus Biologicals; clone 184, epitope: 700 –749 amino acids of TPX2, Novus Biologicals), phosphoCdk/MAPK substrates (34B2, Cell Signaling), Cy3 conjugated-tubulin (Sigma), p-Aurora A (ab18318, Abcam), and monoclonal TPX2 (18D5-1, Abcam) were used for Western blots, immunoprecipitations, and/or immunofluorescence experiments.

Techniques: Staining, Blocking Assay, Expressing, Transfection

Journal: Journal of Biological Chemistry

Article Title: Phosphorylation of Targeting Protein for Xenopus Kinesin-like Protein 2 (TPX2) at Threonine 72 in Spindle Assembly

doi: 10.1074/jbc.m114.591545

Figure Lengend Snippet: FIGURE 6. Effects of GFP-TPX2 T72A on the polarity of mitotic spindles in HeLa cells with or without endogenous TPX2. A, representative photographs of mitotic HeLa cells at prometaphase and metaphase with monopolar, bipolar, and multipolar mitotic spindle poles. Scale bar, 10 m. B, Western blots showing the levels of endogenous TPX2, GFP-TPX2 WT, and GFP-TPX2 T72A in cells with intact levels of TPX2. C, bar graphs showing the number of cells with different mono-, bi-, or multipolar mitotic spindles in each group. Cells with mitotic spindles were fixed and stained with Cy3-conjugated tubulin for MT visualization. GFP-TPX2 T72A expression results in a significant increase in the percentage of cells with multipolar spindles in the presence of endogenous TPX2. ANOVA comparing the three groups shows high significance with p 0.001. Neuman-Keuls test was used to compare each group: GFP (1.49 0.47) versus T72A (12.72 2.10), p 0.001; TPX2 WT (3.36 0.40) versus T72A (12.72 2.10), p 0.001; group (mean S.E.); ***, p 0.001; NS, not significant (GFP versus TPX2). At least 100 cells for each set of experiments were used for quantification, 5 independent experiments were performed. Error bars indicate S.E. D, Western blots showing the levels of endogenous TPX2, GFP-TPX2 WT, and GFP-TPX2 T72A in HeLa cells co-transfected with GFP-vector, GFP-TPX2 WT, or GFP-TPX2T72AtogetherwithTPX2siRNAtargetingthe3UTRofTPX2mRNA.E,bargraphsshowingthenumberofcellswithdifferentmono-,bi-,ormultipolar mitotic spindles in each group. Cells with mitotic spindles were fixed and stained with Cy3-conjugated tubulin for MT visualization. Knockdown of TPX2 in GFP-transfected cells results in a significant 5.4% increase in multipolar spindles versus control cells without TPX2 depletion. GFP-TPX2 T72A expression produces an even greater 9.8 and 7.5% increase in the percentage of cells with multipolar spindles when compared with GFP/TPX2 siRNA and GFP-TPX2 WT/TPX siRNA, respectively. n 3, ANOVA test was used the compare the four groups (p 0.01). The Neuman-Keuls test was used to compare the following groups: control (with control siRNA) (2.43 0.41) versus GFP (7.94 1.5), p 0.05; GFP (7.94 1.5) versus TPX2 WT (10.13 1.2), NS; WT (10.13 1.2) versus T72A (17.67 3.2), p 0.05; GFP (7.94 1.5) versus T72A (17.67 3.2), p 0.05; group (mean S.E.); *, p 0.05; NS, not significant. n at least 500 cells for each set of experiments; 3 independent experiments were performed. Error bars indicate S.E.

Article Snippet: Antibodies—Primary Abs against Cdk1, Cdk2 (Santa Cruz), Thr(P)72 TPX2 (homemade, described above), Cyclin B (Abcam), 9124 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 290 • NUMBER 14 • APRIL 3, 2015 at H A R V A R D U N IV E R SIT Y on A pril 15, 2015 http://w w w .jbc.org/ D ow nloaded from -actin (Chemicon), TPX2 (clone 183, epitope: 150 –200 amino acids of human TPX2, Novus Biologicals; clone 184, epitope: 700 –749 amino acids of TPX2, Novus Biologicals), phosphoCdk/MAPK substrates (34B2, Cell Signaling), Cy3 conjugated-tubulin (Sigma), p-Aurora A (ab18318, Abcam), and monoclonal TPX2 (18D5-1, Abcam) were used for Western blots, immunoprecipitations, and/or immunofluorescence experiments.

Techniques: Western Blot, Staining, Expressing, Transfection, Plasmid Preparation, Knockdown, Control

Journal: Journal of Biological Chemistry

Article Title: Phosphorylation of Targeting Protein for Xenopus Kinesin-like Protein 2 (TPX2) at Threonine 72 in Spindle Assembly

doi: 10.1074/jbc.m114.591545

Figure Lengend Snippet: FIGURE 7. Overactivation of Aurora A and increased spindle length, a measure of Eg5 activity, in TPX2 T72A-expressing cells. A–C show 293 mitotic cells (prometaphase/metaphase) previously transfected with GFP-TPX2 WT (WT) or GFP-TPX2 T72A (T72A) expression vectors. A, representative photographs of WT- and T72A-transfected cells stained for Thr(P)288, a phosphoresidue indicative of the activity of Aurora kinase A. Dotted circles identify the poles. Scatter plots show the P-Aurora signal at centrosomes relative to total GFP signal. GFP-TPX2 T72A induces higher Aurora A activity than GFP-TPX2 WT (GFP-TPX2 WT (0.07 0.01, n 13) versus GFP-TPX2 T72A (0.14 0.03, n 18); *, p 0.05 by t test). B, representative photographs of the spindle length detected in mitotic 293 cells transfected with GFP-TPX2 WT or T72A. Scatter plots show the spindle length in both groups. T72A- expressing cells display longer spindles than WT-expressing cells (GFP-TPX2 WT (58.95 2.12, n 18) versus GFP-TPX2 T72A (66.67 2.20, n 21); **, p 0.01 by t test). C, representative images of the -tubulin signal detected in GFP-TPX2 WT and T72A-trasnfected 293 cells. No significant difference was detected between these two groups (GFP-TPX2 WT (1.93 0.41, n 15) versus GFP-TPX2 T72A (1.97 0.49, n 13); NS, non significant by t test). In all the panels: the group is the mean S.E.. Scale bar, 10 m.

Article Snippet: Antibodies—Primary Abs against Cdk1, Cdk2 (Santa Cruz), Thr(P)72 TPX2 (homemade, described above), Cyclin B (Abcam), 9124 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 290 • NUMBER 14 • APRIL 3, 2015 at H A R V A R D U N IV E R SIT Y on A pril 15, 2015 http://w w w .jbc.org/ D ow nloaded from -actin (Chemicon), TPX2 (clone 183, epitope: 150 –200 amino acids of human TPX2, Novus Biologicals; clone 184, epitope: 700 –749 amino acids of TPX2, Novus Biologicals), phosphoCdk/MAPK substrates (34B2, Cell Signaling), Cy3 conjugated-tubulin (Sigma), p-Aurora A (ab18318, Abcam), and monoclonal TPX2 (18D5-1, Abcam) were used for Western blots, immunoprecipitations, and/or immunofluorescence experiments.

Techniques: Activity Assay, Expressing, Transfection, Staining

Journal:

Article Title: NMR insights into a megadalton-size protein self-assembly

doi: 10.1110/ps.035840.108

Figure Lengend Snippet: (A) Histogram of distribution of hydrodynamic radii obtained from “regularization analysis” of data from dynamic light scattering experiments for 100 μM GED in 0.1 M phosphate buffer containing 1 mM EDTA, 150 mM NaCl at pH 5.7; average R h = 22.37 nm. (B) Plot of estimated molecular weight vs. hydrodynamic radii for 18 different proteins from literature (Claes et al. 1992); dashed line extrapolates the R h value of 22.37 nm on the fitted line (solid line) and corresponds to a molecular weight of ∼5 MDa. (C) Plot of estimated molecular weight vs. known molecular weight for the 18 proteins used in plot B; correlation coefficient between the two is found to be 0.99. (D) Size-exclusion chromatogram of GED (100 μM, 100 μL) in 0.1 M phosphate buffer containing 1 mM EDTA, 150 mM NaCl at pH 5.7, run on a Superose 6 10/300 GL column (GE Healthcare) using the BioLogic FPLC system (Bio-Rad), at a flow rate of 0.5 mL/min.

Article Snippet: Size-exclusion chromatography Size-exclusion chromatography was performed using Superose 6 10/300 GL column (GE Healthcare) with 0.1 M phosphate buffer containing 1 mM EDTA, 150 mM NaCl at pH 5.7, at a flow rate of 0.5 mL/min with absorbance monitored at 280 nm using a BioLogic FPLC system (Bio-Rad).

Techniques: Molecular Weight

Journal: Scientific Reports

Article Title: Effect of human very low-density lipoproteins on cardiotrophin-like cytokine factor 1 (CLCF1) activity

doi: 10.1038/s41598-018-22400-y

Figure Lengend Snippet: CLCF1 binds recombinant and serum ApoE. ( A ) Recombinant CLCF1 (200 ng) and ApoE2, 3 or 4 (250 ng) were incubated alone or in combination for 16 h. The samples were subjected to immunoprecipitation (IP) with anti-CLCF1 and protein G agarose. Immunoprecipitated proteins were analyzed by WB using mAbs specific for ApoE (upper panel) or CLCF1 (lower panel). Lanes “CLC 2 ng” and “ApoE3 20 ng”, show results of recombinant proteins directly subjected to SDS-PAGE and WB blot analysis. ( B ) CLCF1 and ApoE form a complex when co-expressed in HEK-293 cells. The cell culture medium from stable transfectants expressing the indicated proteins in combination with CRLF1 or from cells transfected with empty expression vector (lane D5) was subjected to immunoaffinity chromatography with rat-anti-human ApoE and anti-rat Ig Agarose. The eluates were analysed by WB using anti-ApoE or anti-CLC mAbs (upper panels). Supernatants of the stable transfectants were analyzed for the presence of ApoE or CLCF1 by WB (lower panels). ( C and D ) CLCF1 binds mouse and human serum ApoE. ( C ) Mouse serum samples isolated from WT or ApoE −/− mice were diluted 1:20, mixed with biotinylated mouse CLCF1 and the complexes immunoprecipitated with rabbit anti-mouse ApoE and anti-rabbit Ig agarose. The eluates were analysed for ApoE or CLCF1 by WB using anti-mouse ApoE or HRP-labelled streptavidin. The last two lanes show the signals obtained with 40 ng of biotinylated mouse CLCF1 or ApoE included as control. ( D ) Human serum samples were diluted 1:20, mixed with biotinylated human CLCF1 and the complexes immunoprecipitated with rat anti-ApoE and anti-rat Ig agarose. The eluates were analyzed for ApoE or CLCF1 by WB using anti-ApoE or HRP-labelled streptavidin. The last two lanes show the signals obtained with 40 ng of biotinylated human CLCF1 or ApoE included as control. Blots’ images where cropped to show relevant areas.

Article Snippet: Eluates were analyzed by WB using HRP-streptavidin (GE-Healthcare Life Science, Cedarlane, Burlington, ON) or rabbit anti-mouse ApoE Ab (Abcam Inc, Cedarlane) followed by HRP-labelled TrueBlot™ anti-rabbit Ab (Rockland Immunochemicals Inc).

Techniques: Recombinant, Incubation, Immunoprecipitation, SDS Page, Cell Culture, Expressing, Transfection, Plasmid Preparation, Chromatography, Isolation

Journal: Scientific Reports

Article Title: Effect of human very low-density lipoproteins on cardiotrophin-like cytokine factor 1 (CLCF1) activity

doi: 10.1038/s41598-018-22400-y

Figure Lengend Snippet: AlphaLISA proximity assay shows CLCF1-ApoE complex formation that is increased by the lipid DMPC. ( A ) ApoE2, 3 or 4 were mixed with CLCF1 (10 nM) in PBS 0.1% BSA for 1 h with IgG rat anti-ApoE (3 nM) and anti-IgG rat acceptor beads (10 μg/mL). Streptavidin donor beads (40 μg/mL) were further added for 1 h and 615 nm fluorescence signal assessed. ( B ) The indicated ApoE isotypes (30 nM) were mixed at 37 °C with increasing amount of DMPC for 1 h. ApoE-DMPC mix were further incubated with CLCF1 and subjected to AlpahLISA proximity assay. Histograms indicate the mean fluorescence intensity of 3 independent samples ± SD.

Article Snippet: Eluates were analyzed by WB using HRP-streptavidin (GE-Healthcare Life Science, Cedarlane, Burlington, ON) or rabbit anti-mouse ApoE Ab (Abcam Inc, Cedarlane) followed by HRP-labelled TrueBlot™ anti-rabbit Ab (Rockland Immunochemicals Inc).

Techniques: Proximity Assay, Fluorescence, Incubation

Journal: Scientific Reports

Article Title: Effect of human very low-density lipoproteins on cardiotrophin-like cytokine factor 1 (CLCF1) activity

doi: 10.1038/s41598-018-22400-y

Figure Lengend Snippet: ApoE3 increases CLCF1-induced CNTFR activation. ( A ) Ba/F3 transfected with CNTFRα, gp130 and LIFRβ cDNA were successively incubated with mouse biotinylated CLCF1 (1 μg/ml) alone or in the presence of ApoE3 (10 mg/ml) and PE-labelled streptavidin and fluorescence was assessed by flow cytometry. Vehicle: cells incubated with PE-streptavidin alone. ( B ) Ba/F3-CNTFR were stimulated with CLCF1 (100 ng/ml) or ApoE3 (10 mg/ml) alone or in combination for 15 min. STAT3 tyrosine phosphorylation was assessed by intracellular staining with anti-pSTAT3 mAb and flow cytometry. Vehicle: cells incubated with PE-streptavidin alone. Histograms indicate the mean fluorescence intensity of 3 independent samples ± SD.

Article Snippet: Eluates were analyzed by WB using HRP-streptavidin (GE-Healthcare Life Science, Cedarlane, Burlington, ON) or rabbit anti-mouse ApoE Ab (Abcam Inc, Cedarlane) followed by HRP-labelled TrueBlot™ anti-rabbit Ab (Rockland Immunochemicals Inc).

Techniques: Activation Assay, Transfection, Incubation, Fluorescence, Flow Cytometry, Staining

Journal: Scientific Reports

Article Title: Effect of human very low-density lipoproteins on cardiotrophin-like cytokine factor 1 (CLCF1) activity

doi: 10.1038/s41598-018-22400-y

Figure Lengend Snippet: CLCF1 co-purifies or co-elutes with serum lipoprotein complexes. ( A ) WT mouse, ( B ) human or ( C ) ApoE −/− mouse sera were mixed with corresponding biotinylated CLCF1. The mixtures were fractionated by FPLC using a Superose 6 HR 10/ column in order to obtain 40 fractions. The fractions were analyzed for ApoE or CLCF1 by WB using anti-ApoE or HRP-labelled streptavidin. The right lanes show the signals obtained with 40 ng of ApoE or biotinylated CLCF1 included as control. Blots’ images where cropped to show relevant areas.

Article Snippet: Eluates were analyzed by WB using HRP-streptavidin (GE-Healthcare Life Science, Cedarlane, Burlington, ON) or rabbit anti-mouse ApoE Ab (Abcam Inc, Cedarlane) followed by HRP-labelled TrueBlot™ anti-rabbit Ab (Rockland Immunochemicals Inc).

Techniques:

Journal: Scientific Reports

Article Title: Effect of human very low-density lipoproteins on cardiotrophin-like cytokine factor 1 (CLCF1) activity

doi: 10.1038/s41598-018-22400-y

Figure Lengend Snippet: Biotinylated LDL and VLDL bind CLCF1. ( A ) Interaction between CLCF1 and lipoproteins was assessed using ligand blotting assays: 0.1–0.2 μg of hCLC, or 1 μg of BSA were subjected to non-reducing SDS-PAGE. Proteins were revealed by Coomassie blue staining (left section) or electro-transferred to nitrocellulose and incubated successively with 30 μg/ml biotinylated VLDL, LDL or HDL and HRP-labeled neutravidin (middle 3 panels). Signal was revealed by chemiluminescence. Image in the far right section shows results of incubation with HRP-labeled neutravidin alone. Lane hCLC biot; 40 ng of biotinylated CLC was used as positive control for neutravidin detection. Blots’ images where cropped to show relevant areas. ( B ) The formation of LDL and VLDL complexes can be detected using an AlphaLISA proximity assay. Biotinylated hCLCF1 was mixed with LDL, HDL and VLDL covalently coupled to acceptor beads (10 μg/mL). Streptavidin donor beads (40 μg/mL) were further added for 1 h and 615 nm fluorescence signal assessed. Histograms indicate the mean fluorescence intensity of 3 independent samples ± SD.

Article Snippet: Eluates were analyzed by WB using HRP-streptavidin (GE-Healthcare Life Science, Cedarlane, Burlington, ON) or rabbit anti-mouse ApoE Ab (Abcam Inc, Cedarlane) followed by HRP-labelled TrueBlot™ anti-rabbit Ab (Rockland Immunochemicals Inc).

Techniques: SDS Page, Staining, Incubation, Labeling, Positive Control, Proximity Assay, Fluorescence

Journal: Scientific Reports

Article Title: Effect of human very low-density lipoproteins on cardiotrophin-like cytokine factor 1 (CLCF1) activity

doi: 10.1038/s41598-018-22400-y

Figure Lengend Snippet: VLDL increases CLCF1 binding to CNTFR-expressing cells. Ba/F3 transfected with CNTFRα, gp130 and LIFRβ cDNA, IMR32 neuroblastoma or 3T3-L1 pre-adipocytes were successively incubated with species-matched biotinylated CLCF1 (1 μg/ml) alone or in the presence of LDL, HDL or VLDL (all at 100 μg/ml). Cells were then incubated with PE-conjugated streptavidin and fluorescence was assessed by flow cytometry. Vehicle: cells incubated with PE-streptavidin alone. Histograms indicate the mean fluorescence intensity of 3 independent samples ± SD.

Article Snippet: Eluates were analyzed by WB using HRP-streptavidin (GE-Healthcare Life Science, Cedarlane, Burlington, ON) or rabbit anti-mouse ApoE Ab (Abcam Inc, Cedarlane) followed by HRP-labelled TrueBlot™ anti-rabbit Ab (Rockland Immunochemicals Inc).

Techniques: Binding Assay, Expressing, Transfection, Incubation, Fluorescence, Flow Cytometry

Journal: PLOS Pathogens

Article Title: TfR1 facilitates influenza virus endocytosis and uncoating by interacting with NA and M1 via extracellular and intracellular domains

doi: 10.1371/journal.ppat.1013511

Figure Lengend Snippet: (A) Western blot analysis examining the impact of TfR1 knockdown in A549 cells on IAV (A/WSN/1933 (H1N1)) 12 hours infection (MOI = 0.5). Equal loading was confirmed by detecting GAPDH. (B) Immunofluorescent staining of A549 cells and cells with TfR1 knockdown 4h post -infection (A/WSN/1933 (H1N1)) (MOI = 0.5). (C) Evaluation of TfR1 knockdown’s inhibitory effect on IAV infection (MOI = 0.5) in A549 cells. Viral RNA levels were quantified by RT-qPCR at 12 hours post-infection across multiple strains: A/WSN/1933 (H1N1), A/PR/8/1934 (H1N1), and A/Aichi/2/1968 (H3N2). (D) Western blot characterization investigates the correlation between TfR1 expression levels in different cell lines and their susceptibility to IAV infection (MOI = 0.5). Equal loading was confirmed by detecting GAPDH. (E) Comparative evaluation of sialic acid versus TfR1 as potential receptors in mediating IAV entry. A549 cells transfected with TfR1/vector plasmid were treated with neuraminidase (50 units/mL) or BSA at 37°C for 2 hours before infection with IAV (A/WSN/1933 (H1N1), MOI = 0.5) ( left panel ). Alternatively, IAV was pretreated with neuraminidase or BSA at 37°C for 2 hours before infecting A549 cells ( right panel ). After 12 hours, infected cells were lysed and quantified by RT-qPCR. (F) Comparative evaluation of TfR1 as a potential receptor mediating IAV attachment and endocytosis. A549 and TfR1-knockdown cells were incubated with either unlabeled IAVs (A/WSN/1933 (H1N1), MOI = 10) or sulfo-NHS-SS-Biotin-labeled IAVs (MOI = 5) at 4°C for 1 hour (to allow attachment) or subsequently shifted to 37°C for 2 hours (to allow endocytosis). After washing, attached viruses were quantified by RT-qPCR, and internalized viruses were measured by flow cytometry. (G) Flow cytometry analysis assessing TfR1 involvement in IAV entry, quantifying internalized biotin-SS-labeled IAVs (A/WSN/1933 (H1N1)) (MOI = 5) in A549 cells, TfR1-knockout A549 cells, and CHO cells. TCEP treatment was applied to cleave disulfide bond and remove the biotin tag on non-internalized IAV particles, followed by fixation, permeabilization and incubation with AF488-conjugated streptavidin. Unpaired t-tests were used for statistical analysis with corresponding p-values. *p < 0.05; **p < 0.01; ***p < 0.001; and ****p < 0.0001, while “n.s.” indicates non-significance.

Article Snippet: Transferrin Receptor/TFR1/CD71 Protein, Human, Recombinant (ECD, His Tag), HPLC-verified (Sino Biological, Cat#11020-H07H), Influenza A H1N1 (A/WSN/1933) Hemagglutinin/ HA Protein (His Tag) (Sino Biological, Cat#11692-V08H), Influenza A H1N1 (A/Puerto Rico/8/34/Mount Sinai) Matrix protein 1/ M1 Protein (His Tag), (Sino Biological, Cat#40010-V07E).

Techniques: Western Blot, Knockdown, Infection, Staining, Quantitative RT-PCR, Expressing, Transfection, Plasmid Preparation, Incubation, Labeling, Flow Cytometry, Knock-Out