Journal: Proteomes

Article Title: Mapping the Proteome of the Synaptic Cleft through Proximity Labeling Reveals New Cleft Proteins

doi: 10.3390/proteomes6040048

Figure Lengend Snippet: SynCAM 1-peroxidase fusion protein peroxidase-mediated proximity labeling in the synaptic cleft. ( A ) APEX2 or HRP (image RCSB PDB [ , ] ( www.rcsb.org ) of PDB ID 1HCH ) peroxidase was inserted at the base of the SynCAM 1 extracellular domain, with immunoglobulin (Ig) domains, trans-membrane (TM) region, and intracellular PDZ domain interaction sequence indicated. APEX2 or HRP catalyzes the formation of a short-lived biotin-AEEA-phenoxyl radical (red dot) after exogenous addition of H 2 O 2 and membrane-impermeable biotin-AEEA-phenol (blue dot). ( B ) Exogenous biotin-AEEA-phenol induced biotinylation only at the cell surface. Staining for biotin (visualized by StreptAvidin-Alexa488) in HEK293T cells expressing SynCAM 1-APEX2 in presence (+) but not in absence (−) of H 2 O 2 . ( C ) Exogenous biotin-AEEA-phenol did not induce biotinylation in HEK293T cells expressing cytosolic APEX2-NES.

Article Snippet: For dSTORM imaging of SynCAM 1-HRP, cultured neurons on coverslips were transfected at 18 div using lipofectamine 2000 (1 µg/µL DNA) (Thermo Fisher Scientific, Waltham, MA, USA) and 1 µg/coverslip total pAAV SynCAM 1-HRP DNA in 50 µL opti-mem (Thermo Fisher Scientific) per coverslip.

Techniques: Labeling, Sequencing, Staining, Expressing

Journal: Proteomes

Article Title: Mapping the Proteome of the Synaptic Cleft through Proximity Labeling Reveals New Cleft Proteins

doi: 10.3390/proteomes6040048

Figure Lengend Snippet: Synaptic SynCAM 1-HRP expression and biotinylation. ( A ) Two-color dSTORM reconstruction of synapses from 21 days-in-vitro (div) rat hippocampal neurons surface-labeled by immunostaining with anti-FLAG antibodies against exogenous SynCAM 1-HRP containing the FLAG epitope (magenta) and the endogenous excitatory postsynaptic marker Homer (cyan). Top left, overview. Enlarged panels show one synapse with SynCAM 1-HRP and Homer localizations fit by a convex hull to demarcate the PSD border (dotted outline). SynCAM 1-HRP localizations are at the periphery of the PSD. Diagonal line, line scan used for ( B ). See for additional examples and quantitative analyses. ( B ) Protein localization distribution perpendicular to the trans-synaptic axis. Densities were determined by dSTORM and normalized to the peak of each channel and measured over the distance shown in A. ( C ) Model of HRP targeting by the reporter SynCAM 1-HRP to excitatory synaptic clefts for biotinylation of proximal surface proteins. ( D ) Following the proximity labeling reaction with membrane-impermeant biotin-AEEA-phenol, biotin staining was detected in SynCAM 1-HRP transduced rat cortical neurons along dendrites at excitatory synaptic sites visualized by immunostaining of Homer (arrows). ( E ) Targeting of HRP by the reporter Membrane-HRP to the plasma membrane of dendrites. ( F ) Staining for biotin was detected in Membrane-HRP transduced rat cortical neuronal cultures along dendrites after proximity labeling with biotin-AEEA-phenol.

Article Snippet: For dSTORM imaging of SynCAM 1-HRP, cultured neurons on coverslips were transfected at 18 div using lipofectamine 2000 (1 µg/µL DNA) (Thermo Fisher Scientific, Waltham, MA, USA) and 1 µg/coverslip total pAAV SynCAM 1-HRP DNA in 50 µL opti-mem (Thermo Fisher Scientific) per coverslip.

Techniques: Expressing, In Vitro, Labeling, Immunostaining, FLAG-tag, Marker, Staining

Journal: Proteomes

Article Title: Mapping the Proteome of the Synaptic Cleft through Proximity Labeling Reveals New Cleft Proteins

doi: 10.3390/proteomes6040048

Figure Lengend Snippet: Scaled proximity labeling in cultured neurons. ( A ) Rat cortical neurons were transduced with rAAV encoding HRP reporters (orange) at 14 div and underwent proximity labeling at 21 div. SynCAM 1-HRP (orange) biotinylates synaptic proteins that are purified and digested for LFQ. ( B ) Each biological replicate included samples from the 5 conditions shown. ( C ) Western Blot of samples visualized by NeutrAvidin-DyLight488. Endogenously biotinylated proteins (arrow heads) are marked and weight ranges (brackets) wherein exogenously biotinylated proteins were detected are indicated. ( D ) Example comparisons of molar abundance ranges. Four biological replicates were performed and molar abundance (relative iBAQ or riBAQ) of all detected proteins in SynCAM 1-HRP samples were compared. Graphs show pair-wise comparisons of the 1st biological replicate with the 2nd, 3rd, and 4th replicate, respectively. Endogenously biotinylated proteins (histones, mitochondrial carboxylases), cytosolic proteins (GAPDH, Erlin-2) and synaptic cleft proteins (Contactin-1, Kilon, Hapln1, Noelin-1, Latrophilin-3, Neurexin-1, Neuroligin-1) are indicated.

Article Snippet: For dSTORM imaging of SynCAM 1-HRP, cultured neurons on coverslips were transfected at 18 div using lipofectamine 2000 (1 µg/µL DNA) (Thermo Fisher Scientific, Waltham, MA, USA) and 1 µg/coverslip total pAAV SynCAM 1-HRP DNA in 50 µL opti-mem (Thermo Fisher Scientific) per coverslip.

Techniques: Labeling, Cell Culture, Transduction, Purification, Western Blot

Journal: Proteomes

Article Title: Mapping the Proteome of the Synaptic Cleft through Proximity Labeling Reveals New Cleft Proteins

doi: 10.3390/proteomes6040048

Figure Lengend Snippet: Data filtering based on proteins of known sub-cellular localizations. ( A ) Detected proteins were sorted into 4 categories based upon literature and these categories were used in Filter 1 and 2. Filter 3 used the number of biological replicates a protein passed Filter 1 and 2. ( B ) Histograms of the extent of biotinylation by SynCAM 1-HRP vs. no reporter control for Filter 1 (left plot) and vs. Mem-HRP for Filter 2 (right plot). Cut-off values determined in ( C ) for Filter 1 and Filter 2 are indicated. ( C ) Receiver Operating Characteristic (ROC) curve analysis to determine optimal enrichment for Filter 1 (left plot) or Filter 2 (right plot) by plotting the True Positive Rate (TPR: known synaptic proteins) minus the False Positive Rate (FPR). FPR1: known intracellular proteins; FPR2: known membrane proteins. At maximum (TPR-FPR), the log 2 value is determined at the corresponding protein in the ranked protein list, which served as a cut-off value in ( B ). ( D ) Enrichment for excitatory synaptic cleft proteins through filtering. All proteins combined in four biological replicate experiments were subjected to Filter 1, Filter 2, and Filter 3. A final manual curation step removed remaining false positives. ( E ) Relative enrichment per group of proteins of known sub-cellular localization. Per group, all proteins (100% per group) are depicted as a circle of unitary size. Each filter step reduces protein number and circle area proportionally. Note that after Filter 1, the total proteins in group of known synaptic proteins is 100% and area of circle is unitary. ( F ) ROC analysis of TPR-FPR2 for Filter 3. Indicated are proteins that passed Filters 1 and 2 four times (red), three times (purple), two times (yellow), and one time (green).

Article Snippet: For dSTORM imaging of SynCAM 1-HRP, cultured neurons on coverslips were transfected at 18 div using lipofectamine 2000 (1 µg/µL DNA) (Thermo Fisher Scientific, Waltham, MA, USA) and 1 µg/coverslip total pAAV SynCAM 1-HRP DNA in 50 µL opti-mem (Thermo Fisher Scientific) per coverslip.

Techniques:

Journal: Proteomes

Article Title: Mapping the Proteome of the Synaptic Cleft through Proximity Labeling Reveals New Cleft Proteins

doi: 10.3390/proteomes6040048

Figure Lengend Snippet: Characterization of R-PTP-zeta expression and synaptic markers in vitro. ( A ) Immunostaining for Bassoon, R-PTP-zeta, and SynCAM 1 in cultured neurons. R-PTP-zeta (grey in single color image) and SynCAM 1 (grey in single color image) were stained under non-permeabilizing conditions using antibodies detecting extracellular epitopes and Bassoon (grey in single color image) was stained under permeabilizing conditions. Yellow in larger composite (left) image indicates colocalization of Bassoon (red) and extracellular R-PTP-zeta (green). White in larger composite (right) image indicates colocalization of extracellular SynCAM 1 (magenta) and extracellular R-PTP-zeta (green). Panels below show enlarged dendritic segments from the composite images to visualize colocalization (arrowheads). ( B ) Immunostaining for Homer and R-PTP-zeta and SynCAM 1 in cultured neurons. Extracellular R-PTP-zeta (grey in single color image) and SynCAM 1 (grey in single color image) were immunostained under non-permeabilizing conditions as in ( A ) and Homer (grey in single color image) was immunodetected under permeabilizing conditions. Yellow in larger composite image (left) indicates colocalization of Homer (red) and extracellular R-PTP-zeta (green). White in larger composite image (right) indicates colocalization of extracellular SynCAM 1 (magenta) and extracellular R-PTP-zeta (green). Panels below show enlarged dendrites from the composite images. Arrowheads mark sites of colocalization.

Article Snippet: For dSTORM imaging of SynCAM 1-HRP, cultured neurons on coverslips were transfected at 18 div using lipofectamine 2000 (1 µg/µL DNA) (Thermo Fisher Scientific, Waltham, MA, USA) and 1 µg/coverslip total pAAV SynCAM 1-HRP DNA in 50 µL opti-mem (Thermo Fisher Scientific) per coverslip.

Techniques: Expressing, In Vitro, Immunostaining, Cell Culture, Staining

Journal: Nature Communications

Article Title: PABPN1 gene therapy for oculopharyngeal muscular dystrophy

doi: 10.1038/ncomms14848

Figure Lengend Snippet: ( a ) Three constructs were cloned into pAAV vectors and used in this study: a tricistronic shRNA construct including hairpins sh-1, sh-2 and sh-3 driven each by a different polymerase III promoter (U61, U69 and H1, respectively), the sequence-optimized PABPN1 (optPABPN1) driven by the SPc5-12 promoter and tagged with a MYC-tag, and the human expanded PABPN1 (expPABPN1) driven by the SPc5-12 promoter and tagged with a FLAG-tag. ( b ) HEK293T cells were transfected with 4 μg per well of pAAV-shRNA3X in triplicate with or without AAV plasmids expressing expPABPN1 or optPABPN1. Untransfected cells and cells transfected with AAV plasmid expressing shRNA for HBVpol were used as a control. Seventy-two hours post transfection, samples were collected and PABPN1 was detected by western blot and quantified by densitometric analysis using ImageJ software. Transfection was performed twice. PABPN1 expression in each condition was normalized by GAPDH expression level and then by the value of untransfected cells. Transfection with a plasmid expressing shRNA3X induced efficient PABPN1 knockdown even when expPABPN1 was co-expressed compared to untransfected cells. The co-expression with optPABPN1 restored PABPN1 expression to the normal level. ( c ) Representative image of a western blot used for these analyses. ( d ) FLAG is not detected when samples are transfected with shRNA3X. However, in samples prepared from cells transfected with pAAV-opPABPN1, MYC-Tag is detected by western blot. Detection of MYC-Tag shows that optPABPN1 is resistant to degradation by shRNA3X in vitro . Data are presented as mean±s.e.m., n =4 (shRNA3X+optPABPN1) or n =6 (all the other groups). One-way ANOVA test with Bonferroni post-hoc test * P <0.05, ** P <0.01, NS, not significant.

Article Snippet: Membrane was stained with primary antibodies raised against PABPN1 (Abcam rabbit monoclonal, ab75855, 1/10,000, overnight (ON)), Vinculin (Sigma-Aldrich mouse monoclonal SAB4200080, 1/10,000, ON), GAPDH (Abcam mouse monoclonal ab9485, 1/2,500, ON), MYC-tag (Abcam rabbit polyclonal, ab9106, 1/5,000, ON) or FLAG (F3165 Sigma monoclonal, 1/2,000 ON).

Techniques: Construct, Clone Assay, shRNA, Sequencing, FLAG-tag, Transfection, Expressing, Plasmid Preparation, Western Blot, Software, In Vitro

Journal: Cell Death & Disease

Article Title: Dominant-negative ATF5 rapidly depletes survivin in tumor cells

doi: 10.1038/s41419-019-1872-y

Figure Lengend Snippet: a Transfection with GFP-FLAG-dn-ATF5 depletes survivin in PC3 prostate tumor cells. Cells were transfected as described in methods and assessed 3 days later for relative survivin levels (normalized to ACTIN) by western immunoblotting. Left panel shows representative blot, right panel shows quantification from 3 independent experiments. b Examples of survivin localization in T98G cells. Transfected cultures were immunostained for survivin and co-stained with Hoechst 33828 to visualize nuclear DNA. Blue/white arrowhead shows a cell without significant survivin expression. Red and blue arrowheads show cells with both cytoplasmic (red arrowheads) and nuclear (blue arrowheads) staining. Yellow arrowhead shows a cell with nuclear-only staining. Scale bar = 10 µm. c , d Transfection with GFP-FLAG-dn-ATF5 depletes nuclear-only localized survivin and increases the proportion of cells without detectable survivin expression in T89G and LN229 cultures at 24 h (C) or at 3–5 d (D). Transfected cells (GFP+) were blindly scored for survivin localization using criteria described in panel b . Data are from three independent experiments, each carried out in triplicate. In each experiment about 200–300 cells were evaluated. e Transfection with GFP-FLAG-dn-ATF5 does not increase the incidence of apoptosis in cultures of T89G and LN229 cultures at 24 h, a time when survivin is already depleted. Transfected cells (GFP+) in cultures described in panels c and d were scored for proportion with apoptotic nuclei. Data are from three independent experiments, each carried out in triplicate. In each experiment about 200–300 cells were evaluated. f Transfection with GFP-FLAG-dn-ATF5 promotes cell death at 3–5 days. As in e , but at 5 days for T98G and 3 days for LN229 cells.

Article Snippet: FLAG-tagged human survivin cDNA cloned into a pCMV6-entry vector was obtained from Origene, Rockville MD (#RC205935).

Techniques: Transfection, Western Blot, Staining, Expressing

Journal: Cell Death & Disease

Article Title: Dominant-negative ATF5 rapidly depletes survivin in tumor cells

doi: 10.1038/s41419-019-1872-y

Figure Lengend Snippet: a , b CP-dn-ATF5 depletes survivin in T98G, U87 and LN229 glioblastoma cell lines at 24 h after treatment. a Representative western blots. b Data are from three independent experiments. c CP-dn-ATF5 depletes survivin in T98G, U87 and LN229 glioblastoma cell lines at 48 and 72 h after treatment. Normalized survivin to ACTIN ratios are shown for each lane. d Treatment with CP-dn-ATF5 causes a rapid depletion of survivin protein in T98G cultures. Normalized survivin to ACTIN ratios are shown for each lane. e Cell death caused by CP-dn-ATF5 (100 µM) in T98G cultures occurs after survivin depletion. Relative numbers of cells per culture were determined at the indicated times. Three replicate cultures were evaluated at each time point

Article Snippet: FLAG-tagged human survivin cDNA cloned into a pCMV6-entry vector was obtained from Origene, Rockville MD (#RC205935).

Techniques: Western Blot

Journal: Cell Death & Disease

Article Title: Dominant-negative ATF5 rapidly depletes survivin in tumor cells

doi: 10.1038/s41419-019-1872-y

Figure Lengend Snippet: a – d CP-dn-ATF5 depletes survivin mRNA in multiple cancer cell lines at 48 h of treatment. Data are from three independent experiments. e CP-dn-ATF5 (100 µM) causes rapid depletion of survivin mRNA in T98G cells. Values are from one experiment carried out in triplicate

Article Snippet: FLAG-tagged human survivin cDNA cloned into a pCMV6-entry vector was obtained from Origene, Rockville MD (#RC205935).

Techniques:

Journal: Cell Death & Disease

Article Title: Dominant-negative ATF5 rapidly depletes survivin in tumor cells

doi: 10.1038/s41419-019-1872-y

Figure Lengend Snippet: a CP-dn-ATF5 decreases the expression of exogenous FLAG-survivin. Indicated cell lines were infected with lentivirus expressing FLAG-survivin and 48 h later were treated with 100 µM CP-dn-ATF5 for 72 h and then assessed for relative (to ACTIN) FLAG-survivin levels by western immunoblotting. Normalized survivin to ACTIN ratios are shown for each lane. b CP-dn-ATF5 accelerates the turnover of survivin protein in T98G cells. Replicate cultures were treated for 24 h with or without (Control) 100 µM CP-dn-ATF5 and then exposed to 50 µM cycloheximide for the indicated times in the continued presence or absence of CP-dn-ATF5 and then assessed for relative survivin levels by western immunoblotting. c CP-dn-ATF5 accelerates the turnover of survivin protein in T98G cells. Cultures were treated as in b and relative survivin expression determined vs ACTIN and normalized to the zero time value in each independent experiment. Numbers of independent experiments for various points are as follows: Control, 1 h, n = 5; 2 h, n = 6; 3 h, n = 5; 4 h, n = 6; 12 h, n = 1; 24 h, = 5. CP-dn-ATF5: 1 h, n = 5; 2 h, n = 6; 3 h, n = 5; 4 h, n = 5; 12 h, n = 1, 24 h, n = 6. Mean data points were fitted to an exponential curve

Article Snippet: FLAG-tagged human survivin cDNA cloned into a pCMV6-entry vector was obtained from Origene, Rockville MD (#RC205935).

Techniques: Expressing, Infection, Western Blot

Journal: Cell Death & Disease

Article Title: Dominant-negative ATF5 rapidly depletes survivin in tumor cells

doi: 10.1038/s41419-019-1872-y

Figure Lengend Snippet: a Proteasomal inhibitor epoxomicin suppresses loss of survivin expression promoted by CP-dn-ATF5. T98G cells stably expressing FLAG-survivin were treated for 100 µM CP-dn-ATF5 for 24 h in presence or absence (Control, CTR) of 10 nM epoxomicin (EPOX) and assessed by western immunoblotting for levels of FLAG-survivin and ACTIN. Left panel shows representative Western immunoblot. Right panel shows quantification of relative FLAG-survivin levels under each condition. Data are from three independent experiments. b Knockdown of USP9X reduces levels of survivin protein in T98G cells. Cultures were transfected with control (CTR) or USP9X siRNA for 72 h and assessed 3 days later for USP9X, survivin and ACTIN protein levels by western immunoblotting (left panel). Right panel shows relative survivin protein levels for five independent experiments. c USP9X knockdown does not affect survivin mRNA levels in T98G cells. Cultures were treated as in b and assessed for relative survivin mRNA levels. Data are from 3 independent experiments

Article Snippet: FLAG-tagged human survivin cDNA cloned into a pCMV6-entry vector was obtained from Origene, Rockville MD (#RC205935).

Techniques: Expressing, Stable Transfection, Western Blot, Transfection

Journal: Cell Death & Disease

Article Title: Dominant-negative ATF5 rapidly depletes survivin in tumor cells

doi: 10.1038/s41419-019-1872-y

Figure Lengend Snippet: a CP-dn-ATF5 reduces expression of over-expressed FLAG-survivin, but to levels far in excess of endogenous survivin. T98G cultures were infected with lentivirus expressing either pLVX-EF1α-IRES-mCherry (pLV in Figure) or pLVX-EF1α-FLAG-survivin-IRES-mCherry (pLV-survivin in Figure) as indicated and 24 h later treated with or without 100 µM CP-dn-ATF5 for 3 d and assessed for exogenous and endogenous survivin by western immunoblotting with anti-survivin. Long exposure shows levels of endogenous survivin. b Survivin over-expression does not rescue the effects of CP-dn-ATF5 on number or appearance of T98G cells. Cultures were infected with lentiviruses described in panel a as indicated and 24 h later were treated with 100 µM CP-dn-ATF5 for 3 days. Panels show immunofluoresence for mcherry (red) or phase contrast images. Scale bars = 50 µm. c Survivin over-expression does not rescue T98G cells from apoptotic death promoted by CP-dn-ATF5. Cultures were infected with lentivirus described in panel a and 24 h later were treated with 100 µM CP-dn-ATF5 for 3 days. Cultures were harvested and analyzed for proportion of apoptotic cells by flow cytometry. Left panel shows the representative cytometry data and right panel shows quantitative results from three independent experiments, each in triplicate. d Survivin over-expression does not rescue cell number in T98G cultures treated with CP-dn-ATF5. Cultures were infected with above described lentiviruses and 24 h later were treated with or without 100 µM CP-dn-ATF5 as indicated for 3 days. Cultures were harvested and analyzed for total cell numbers. Data are from three independent experiments, each in triplicate. e Survivin over-expression does not rescue promotion of apoptosis by FLAG-GFP-dn-ATF5 in multiple cancer cell lines. Indicated cell lines were co-transfected as shown with a ratio of pLVX:pLE constructs of 3:1 and transfected (GFP+) cells were assessed for proportion with apoptotic nuclei 3 days later. Data are from three independent experiments

Article Snippet: FLAG-tagged human survivin cDNA cloned into a pCMV6-entry vector was obtained from Origene, Rockville MD (#RC205935).

Techniques: Expressing, Infection, Western Blot, Over Expression, Flow Cytometry, Cytometry, Transfection, Construct

Journal: Cell Death & Disease

Article Title: Dominant-negative ATF5 rapidly depletes survivin in tumor cells

doi: 10.1038/s41419-019-1872-y

Figure Lengend Snippet: Proposed role of survivin regulation in the mechanism by which dn-ATF5 kills tumor cells

Article Snippet: FLAG-tagged human survivin cDNA cloned into a pCMV6-entry vector was obtained from Origene, Rockville MD (#RC205935).

Techniques:

Journal: The EMBO Journal

Article Title: A non-canonical role of the inner kinetochore in regulating sister-chromatid cohesion at centromeres

doi: 10.1038/s44318-024-00104-6

Figure Lengend Snippet: ( A ) HeLa cells were transfected with control siRNA or CENP-U siRNA. At 48 h post-transfection, cells were treated with the solvent DMSO or MG132 for 8 h, then cell lysates were immunoblotted with antibodies for Wapl, SMC3, Scc1, and α-Tubulin. ( B – D ) HeLa cells were transfected with control siRNA, CENP-U siRNA, and/or Wapl siRNA. At 48 h post-transfection, cells were treated with MG132 for 8 h, then mitotic chromosome spreads were prepared and stained with the CENP-C antibody and DAPI. The percentage of cells in which the majority of sister chromatids was separated or unseparated was determined in 300 cells for each condition from three independent experiments, with statistics being analyzed for cells with separated chromatids ( B ). Example images are shown ( C ). Cell lysates were immunoblotted with antibodies for Wapl and α-Tubulin ( D ). ( E – H ) HeLa cells were transfected with the indicated siRNAs. At 48 h post-transfection, cells were treated with nocodazole for 3 h. Then mitotic chromosome spreads were prepared and stained with the CENP-C antibody and DAPI. The inter-KT distance was measured on over 1000 chromosomes in 20 cells ( E ). The means and individual data points from two independent experiments are plotted ( F ). Example images are shown ( G ). Cell lysates were immunoblotted with antibodies for Wapl and α-Tubulin ( H ). Data information: Statistics were performed using unpaired Student’s t -test ( B ) and one-way ANOVA ( E ). Means and SDs are shown ( B , E ). Scale bars, 10 µm ( C , G ). NS no significance ( B , E ). .

Article Snippet: Rabbit polyclonal antibodies used were GFP (A11122, Invitrogen), GAPDH (14C10, Cell Signaling Technology/CST), Scc1 (ab992, Abcam), SA2 (Rabbit mAb #5882, CST), SMC1 (A300-055A, Bethyl Laboratories), SMC3 (A300-060A, Bethyl Laboratories), Pds5B (A300-537A, A300-538A, Bethyl Laboratories), GST (G7781, Sigma), Wapl (A300-268A, Bethyl), Sororin (ab192237, Abcam), Flag (GenScript), H2ApT120 (Active motif).

Techniques: Transfection, Control, Solvent, Staining

Journal: The EMBO Journal

Article Title: A non-canonical role of the inner kinetochore in regulating sister-chromatid cohesion at centromeres

doi: 10.1038/s44318-024-00104-6

Figure Lengend Snippet: ( A ) Lysates prepared from asynchronous HeLa cells were subjected to pull-down with GST, GST-Scc1 (281–420), or GST-Scc1 (281–420)-SA2 (80–1060), followed by immunoblotting with antibodies for Wapl and GAPDH, and CBB staining. ( B ) Lysates prepared from HEK-293T cells transiently expressing Wapl-Flag in the forms of WT and the 3xEGE mutant were subjected to pull-down by GST or GST-Scc1 (281–420)-SA2 (80–1060), followed by immunoblotting with the antibody for the Flag-tag, and CBB staining. Irrelevant lanes were removed. ( C ) MBP-Wapl (1–630) in the forms of WT and the 3xEGE mutant were subjected to pull-down by GST, GST-Scc1 (281–420), or GST-Scc1 (281–420) -SA2 (80–1060), followed by immunoblotting with the antibody for MBP, and CBB staining. ( D , E ) U2OS-LacO cells transiently expressing the indicated proteins were stained with antibodies for the Flag-tag and Myc-tag, and DAPI. Example images are shown ( D ). The fluorescence intensity ratio of Wapl-Flag/EGFP at the LacO repeats was quantified in 30 cells for each condition ( E ). ( F , G ) U2OS-LacO cells transiently expressing the indicated proteins were stained with antibodies for the Flag-tag, Myc-tag, and DAPI. Example images are shown ( F ). The fluorescence intensity ratio of SFB-CENP-U/EGFP at the LacO repeats was quantified in 30 cells for each condition ( G ). Data information: The white arrows point to the LacO repeats ( D , F ). Scale bars, 10 µm ( D , F ). Statistics were performed using unpaired Student’s t -test ( E , G ). Means and SDs are shown ( E , G ). .

Article Snippet: Rabbit polyclonal antibodies used were GFP (A11122, Invitrogen), GAPDH (14C10, Cell Signaling Technology/CST), Scc1 (ab992, Abcam), SA2 (Rabbit mAb #5882, CST), SMC1 (A300-055A, Bethyl Laboratories), SMC3 (A300-060A, Bethyl Laboratories), Pds5B (A300-537A, A300-538A, Bethyl Laboratories), GST (G7781, Sigma), Wapl (A300-268A, Bethyl), Sororin (ab192237, Abcam), Flag (GenScript), H2ApT120 (Active motif).

Techniques: Western Blot, Staining, Expressing, Mutagenesis, FLAG-tag, Fluorescence

Journal: The EMBO Journal

Article Title: A non-canonical role of the inner kinetochore in regulating sister-chromatid cohesion at centromeres

doi: 10.1038/s44318-024-00104-6

Figure Lengend Snippet: ( A ) U2OS-LacO cells transiently expressing the indicated proteins and the mutants of EGFP-LacI-Scc1 (I337A/L341A) and Myc-SA2 (W334A) were stained with the antibody for the Myc-tag, and DAPI. Example images are shown. ( B ) U2OS-LacO cells transiently expressing the indicated proteins and the mutants of Myc-SA2 (F367A) and Myc-SA2 (F371A) were stained with the antibody for the Myc-tag and DAPI. Example images are shown. ( C ) Lysates prepared from HEK-293T cells transiently expressing Scc1-GFP and/or Myc-SA2 in the forms of WT and the indicated mutants were subjected to pull-down with GST or GST-CENP-U (1–60), followed by immunoblotting with antibodies for GFP and the Myc-tag, and CBB staining. ( D ) Lysates prepared from HEK-293T cells transiently co-expressing Scc1-GFP and Myc-SA2 (WT and the indicated mutants) were subjected to pull-down with GST or GST-CENP-U (1-60), followed by immunoblotting with antibodies for GFP and the Myc-tag, and CBB staining. Irrelevant lanes were removed. ( E ) Lysates prepared from HEK-293T cells transiently expressing Scc1-GFP and/or Myc-SA2 in the forms of WT and the indicated mutants were subjected to pull-down with MBP or MBP-Wapl (1–630), followed by immunoblotting with antibodies for GFP and the Myc-tag, and CBB staining. ( F ) Lysates prepared from HEK-293T cells transiently co-expressing Scc1-GFP and Myc-SA2 (WT and the indicated mutants) were subjected to pull-down with MBP or MBP-Wapl (1–630), followed by immunoblotting with antibodies for GFP and the Myc-tag, and CBB staining. ( G ) Lysates prepared from HEK-293T cells transiently expressing Scc1-GFP and Myc-SA2 were subjected to pull-down with GST-CENP-U (1–60) in the presence of increased amount of eluted MBP-Wapl (1–630) protein, followed by immunoblotting with antibodies for MBP, GFP and the Myc-tag, and CBB staining. ( H ) Lysates prepared from HEK-293T cells transiently expressing Scc1-GFP and Myc-SA2 were subjected to pull-down with GST-CENP-U (1–60) in the presence of increased amounts of eluted MBP-Wapl (1–630) protein (WT or the 3xEGE mutant), followed by immunoblotting with antibodies for MBP and the Myc-tag, and CBB staining. ( I ) Lysates prepared from HEK-293T cells transiently expressing Scc1-GFP and Myc-SA2 were subjected to pull-down with MBP-Wapl (1–630) in the presence of increased amounts of eluted GST-CENP-U (1–60) protein (WT or the ADA mutant), followed by immunoblotting with antibodies for GST, GFP, and the Myc-tag, and CBB staining. Data information: The white arrows point to the LacO repeats ( A , B ). Scale bars, 10 µm ( A , B ). .

Article Snippet: Rabbit polyclonal antibodies used were GFP (A11122, Invitrogen), GAPDH (14C10, Cell Signaling Technology/CST), Scc1 (ab992, Abcam), SA2 (Rabbit mAb #5882, CST), SMC1 (A300-055A, Bethyl Laboratories), SMC3 (A300-060A, Bethyl Laboratories), Pds5B (A300-537A, A300-538A, Bethyl Laboratories), GST (G7781, Sigma), Wapl (A300-268A, Bethyl), Sororin (ab192237, Abcam), Flag (GenScript), H2ApT120 (Active motif).

Techniques: Expressing, Staining, Western Blot, Mutagenesis

Journal: Frontiers in Cell and Developmental Biology

Article Title: Human Cytomegalovirus vMIA Inhibits MAVS Oligomerization at Peroxisomes in an MFF-Dependent Manner

doi: 10.3389/fcell.2022.871977

Figure Lengend Snippet: vMIA-mediated mitochondrial fragmentation is independent on the fission machinery proteins DLP1 and MFF. (A) Immunofluorescence analyses of MEFs MAVS-MITO cells: (a) control cells, (b) DLP1 silenced cells, (c) MFF silenced cells: (a–c) anti-TIM23; (d–f) overexpression of vMIA-Myc: (d) anti-TIM23, (e) anti-Myc, (f) merge image of d and e; (g–i) overexpression of vMIA-Myc in DLP1 silenced cells: (g) anti-TIM23, (h) anti-Myc, (i) merge image of g and h; (j–l) overexpression of vMIA-Myc in MFF silenced cells: (j) anti-TIM23, (k) anti-Myc, (l) merge image of j and k. Bars represent 10 µm. (B,C) Statistical analysis of mitochondrial morphologies upon overexpression of vMIA-Myc in MEFs MAVS-MITO cells in the absence of DLP1 or MFF, respectively. Approximately 600 cells were analysed per condition. Data represents the means ± SEM of three independent experiments analysed using two-way ANOVA with Bonferroni’s multi comparations test (ns = non-significant, ****– p < 0.0001). Error bars represent SEM.

Article Snippet: Rabbit antibodies against MFF (17090-1-AP, ProteinTech, Manchester, UK) 30, Myc-tag (71D10, 2,278, Cell Signalling Technology, Beverly, MA, United States), FLAG epitope (F7425, Sigma-Aldrich, St. Louis, MO, United States), β-Actin (4,967, Cell Signalling, Danvers, MA, United States), PEX14 (GTX129230, GeneTex, CA,United States) and MAVS (A300-782A, Bethyl Laboratories, TX, United States), and mouse antibodies against MAVS (E-3, SC-166583, Santa Cruz Biotechnology, Dallas, TX, United States), p-STAT1 (Y701, BD Biosciences, San Jose, CA, United States), DLP1 (611,113, BD Bioscience, San Jose, CA, United States), PMP70 (SAB4200181, Sigma-Aldrich, St. Louis, MO, United States), COXIV (4,850, Cell Signalling Technology, Beverly, MA, United States) and α-Tubulin (T9026, Sigma-Aldrich, St. Louis, MO, United States) were used for immunoblotting.

Techniques: Immunofluorescence, Control, Over Expression

Journal: Frontiers in Cell and Developmental Biology

Article Title: Human Cytomegalovirus vMIA Inhibits MAVS Oligomerization at Peroxisomes in an MFF-Dependent Manner

doi: 10.3389/fcell.2022.871977

Figure Lengend Snippet: vMIA-induced peroxisomal and mitochondrial fragmentation is independent of MAVS. vMIA does not disrupt STING-MAVS interaction at peroxisomes. (A) Immunofluorescence analyses of MEFs MAVS KO cells: (a, b) peroxisomal and mitochondrial morphologies in control cells: (a) anti-PMP70, (b) anti-TIM23; (c–e) peroxisomal morphology upon overexpression of vMIA-Myc: (c) anti-PMP70, (d) anti-Myc, (e) merge image of c and d; (f, h) mitochondrial morphology upon overexpression of vMIA-Myc: (f) anti-TIM23, (g) anti-Myc, (h) merge image of f and g. Bars represent 10 µm. (B,C) Statistical analysis of peroxisomal or mitochondrial morphologies upon overexpression of vMIA-Myc in MEFs MAVS KO cells, respectively. Approximately 600 cells were analysed per condition. (D) Co-immunoprecipitation analysis of the interaction between overexpressed STING-FLAG and vMIA-Myc in MEFs MAVS-PEX cells. The pull-down was performed using an antibody against MAVS. Western blot was performed with antibodies against FLAG and Myc. IN represents total cell lysate (input), IP represents immunoprecipitation and OUT represents the cell lysate extracted after incubation with the antibody (output). (E) Quantification of the ratio between IP and IN, in the presence or absence of vMIA. Data represents the means ± SEM of three independent experiments, analysed using unpaired T test (ns - non-significant; ***– p < 0.001, ****– p < 0.0001).

Article Snippet: Rabbit antibodies against MFF (17090-1-AP, ProteinTech, Manchester, UK) 30, Myc-tag (71D10, 2,278, Cell Signalling Technology, Beverly, MA, United States), FLAG epitope (F7425, Sigma-Aldrich, St. Louis, MO, United States), β-Actin (4,967, Cell Signalling, Danvers, MA, United States), PEX14 (GTX129230, GeneTex, CA,United States) and MAVS (A300-782A, Bethyl Laboratories, TX, United States), and mouse antibodies against MAVS (E-3, SC-166583, Santa Cruz Biotechnology, Dallas, TX, United States), p-STAT1 (Y701, BD Biosciences, San Jose, CA, United States), DLP1 (611,113, BD Bioscience, San Jose, CA, United States), PMP70 (SAB4200181, Sigma-Aldrich, St. Louis, MO, United States), COXIV (4,850, Cell Signalling Technology, Beverly, MA, United States) and α-Tubulin (T9026, Sigma-Aldrich, St. Louis, MO, United States) were used for immunoblotting.

Techniques: Immunofluorescence, Control, Over Expression, Immunoprecipitation, Western Blot, Incubation

Journal: Frontiers in Cell and Developmental Biology

Article Title: Human Cytomegalovirus vMIA Inhibits MAVS Oligomerization at Peroxisomes in an MFF-Dependent Manner

doi: 10.3389/fcell.2022.871977

Figure Lengend Snippet: vMIA inhibits MAVS oligomerization at peroxisomes and mitochondria. MFF is essential for the vMIA-mediated inhibition of MAVS oligomerization at peroxisomes but not at mitochondria. (A,B) HEK293T cells infected with SeV in the presence or absence of vMIA. Density gradient assay was performed to demonstrate the separation of endogenous MAVS based on its density. 1—7 represent the fractions isolated from the gradient assay, where 1 represents the fraction with lowest density and 7 represents the fraction with highest density. (A) Peroxisome-enriched fraction, (B) Mitochondria-enriched fraction. (C,D) HEK293T cells infected with SeV in the presence or absence of vMIA and in the absence of MFF. Density gradient assay was performed to demonstrate the separation of endogenous MAVS based on its density. 1—7 represent the fractions isolated from the gradient assay, where 1 represents the fraction with lowest density and 7 represents the fraction with highest density. (C) Peroxisome-enriched fraction, (D) Mitochondria-enriched fraction. (A–D) Immunoblots were performed with antibodies against MAVS, Myc-tag, COXIV, PEX14 and PMP70. (E) Whole cell lysates were resolved by SDS-PAGE. SeV infection, and consequential activation of MAVS downstream signalling, was confirmed using anti-p-STAT1. vMIA-Myc overexpression and MFF silencing were also confirmed using anti-Myc and anti-MFF, respectively. Antibody against Actin was used as loading control.

Article Snippet: Rabbit antibodies against MFF (17090-1-AP, ProteinTech, Manchester, UK) 30, Myc-tag (71D10, 2,278, Cell Signalling Technology, Beverly, MA, United States), FLAG epitope (F7425, Sigma-Aldrich, St. Louis, MO, United States), β-Actin (4,967, Cell Signalling, Danvers, MA, United States), PEX14 (GTX129230, GeneTex, CA,United States) and MAVS (A300-782A, Bethyl Laboratories, TX, United States), and mouse antibodies against MAVS (E-3, SC-166583, Santa Cruz Biotechnology, Dallas, TX, United States), p-STAT1 (Y701, BD Biosciences, San Jose, CA, United States), DLP1 (611,113, BD Bioscience, San Jose, CA, United States), PMP70 (SAB4200181, Sigma-Aldrich, St. Louis, MO, United States), COXIV (4,850, Cell Signalling Technology, Beverly, MA, United States) and α-Tubulin (T9026, Sigma-Aldrich, St. Louis, MO, United States) were used for immunoblotting.

Techniques: Inhibition, Infection, Isolation, Western Blot, SDS Page, Activation Assay, Over Expression, Control

Journal: Nature Communications

Article Title: Cep295 is a conserved scaffold protein required for generation of a bona fide mother centriole

doi: 10.1038/ncomms12567

Figure Lengend Snippet: ( a ) Centriolar distribution of Cep295 at different cell cycle stages. HeLa cells were stained with the indicated antibodies. The bottom panels represent quantification of the local signal intensity of centrin (left) and Cep295 (right). The local signal intensity was visualized in the indicated colours (M: mother centriole; D: daughter centriole). The schematic models represent the localization of Cep295, HsSAS-6 and centrin during the cell cycle. Note that HsSAS-6 disappears in late mitosis. In G1/S phase, the centrosomal linker connects the proximal end of two mother centrioles. Scale bar, 1 μm. ( b ) Cep295 localizes to the assembly site of procentrioles in the earliest stage (arrowheads) and forms a ring-like structure. The images were obtained by TCS SP8 HSR system using antibodies against Cep192 (green) and Cep295 (red). Scale bar, 500 nm. ( c ) 3D-SIM images representing top and side views of Cep295 at mother centrioles. GT335 was used as a centriole wall marker. An arrow points to recruitment of Cep295 onto the daughter centriole wall before the glutamylation of centriolar mitrotubules. Scale bar, 400 nm. ( d , e ) The graph shows the signal intensity of Cep295 at the mother centriole along the dotted line in ( c ). For quantification of the diameter, the distances between intensity maxima were measured (mean±s.d.).

Article Snippet: The following primary antibodies were used in this study: Rabbit polyclonal antibodies against Cep295/KIAA1731 (Sigma, HPA038596, IF 1:1000, WB 1:1000), Cep192 (a gift from Laurence Pelletier, IF 1:1000), Cep192 (Bethyl laboratories, A302–324A, WB 1:1000), Cep152 (Bethyl laboratories, A302–480A, IF 1:1000), Cep152 (Bethyl laboratories, A302–479A, WB 1:1000), CP110 (a gift from Brian David Dynlacht, IF 1:500), CP110 (proteintech, 12780–1-AP, IF 1:500), CPAP (a gift from Pierre Gönczy, IF 1:500), CPAP/CENP-J (Proteintech, 11517-1-AP, IF 1:500), STIL (Abcam, ab89314, IF 1:500, WB 1:1000), Cep135 (Abcam, ab196809, IF 1:1000); mouse monoclonal antibodies against centrin-2 (Millipore, 20H5, IF 1:1000), HsSAS-6 (Santa Cruz Bio-technology, Inc., sc-81431, IF 1:500, WB 1:1000), Plk4 (Merck Millipore, clone 6H5, MABC544, IF 1:500), γ-tubulin (GTU88) (Sigma-Aldrich, T5192, IF 1:1000), Polyglutamylation Modification (GT335, mAb) (AdipoGen, AG-20B-0020-C100, IF 1:5000), FLAG-tag (Sigma, F1804, IF 1:1000, WB 1:1000) and α-tubulin (Sigma-Aldrich, DM1A, IF1:1000); goat polyclonal antibody against GFP, FITC-conjugated (Abcam, ab6662, IF 1:300).

Techniques: Staining, Marker

Journal: Nature Communications

Article Title: Cep295 is a conserved scaffold protein required for generation of a bona fide mother centriole

doi: 10.1038/ncomms12567

Figure Lengend Snippet: ( a ) Schematic of Cep295 function described in this figure. ( b ) Three colour staining of centrioles in control and Cep295-depleted cells. The cells were stained with the indicated antibodies. ODF2 was used as an older mother centriole marker. Arrows point to a new mother centriole (M: older mother centriole; NM: new mother centriole). Scale bar, 1 μm. ( c , d ) HeLa cells transfected with control siRNA or Cep295 siRNA for 72 h were stained with indicated antibodies. CP110 and centrin were used as centriole markers . Scale bar, 1 μm. ( c ) The number shown in the bottom panels represents the number of Plk4/HsSAS-6/STIL foci. ( d ) Histograms represent frequency of interphase cells with ≧2 Plk4/HsSAS-6/STIL/CPAP foci in each condition. Values are mean percentages±s.e.m from three independent experiments ( N =30 for each condition). *** P <0.001; ** P <0.01, (two-tailed t -test). ( e ) HeLa cells were treated with control siRNA or Cep295 siRNA, followed by transfection with an empty vector (−) or, pCMV5-Plk4ΔPEST-FLAG wild-type. The cells were stained with the indicated antibodies. An arrow points to the defect in multiple centriole formation induced by PLK4 over-expression, upon Cep295 depletion. Scale bars, 5 μm in the low-magnified view, 1 μm in the inset. Schematic illustrations show frequency of interphase cells with 2 or 1 over-duplicated centriole foci. ( f ) Cep295-depleted HeLa cells were stained with the indicated antibodies. Almost all control cells harbour ⩾2 Cep192 and ⩾2 Cep152 foci per cell, whereas only 12 and 5% of Cep295-depleted cells have ⩾2 Cep192 and ⩾2 Cep152 foci per cell, respectively. Scale bar, 1 μm. ( g , h ) To monitor the expression levels of Cep295 at the mother and daughter centrioles, the triple staining analysis was performed with the indicated antibodies as shown in the representative panels. HeLa cells were transfected with control or Cep295 siRNA for 24 h. ( g ) Arrows point to the defective recruitment of PCM components in the Cep295-depleted cells just after disengagement. Scale bar, 1 μm. ( h ) Histograms represent frequency of late mitotic cells with the indicated category in each condition. Values are mean percentages±s.e.m from three independent experiments ( N =30 for each condition). ** P <0.01 (two-tailed t -test).

Article Snippet: The following primary antibodies were used in this study: Rabbit polyclonal antibodies against Cep295/KIAA1731 (Sigma, HPA038596, IF 1:1000, WB 1:1000), Cep192 (a gift from Laurence Pelletier, IF 1:1000), Cep192 (Bethyl laboratories, A302–324A, WB 1:1000), Cep152 (Bethyl laboratories, A302–480A, IF 1:1000), Cep152 (Bethyl laboratories, A302–479A, WB 1:1000), CP110 (a gift from Brian David Dynlacht, IF 1:500), CP110 (proteintech, 12780–1-AP, IF 1:500), CPAP (a gift from Pierre Gönczy, IF 1:500), CPAP/CENP-J (Proteintech, 11517-1-AP, IF 1:500), STIL (Abcam, ab89314, IF 1:500, WB 1:1000), Cep135 (Abcam, ab196809, IF 1:1000); mouse monoclonal antibodies against centrin-2 (Millipore, 20H5, IF 1:1000), HsSAS-6 (Santa Cruz Bio-technology, Inc., sc-81431, IF 1:500, WB 1:1000), Plk4 (Merck Millipore, clone 6H5, MABC544, IF 1:500), γ-tubulin (GTU88) (Sigma-Aldrich, T5192, IF 1:1000), Polyglutamylation Modification (GT335, mAb) (AdipoGen, AG-20B-0020-C100, IF 1:5000), FLAG-tag (Sigma, F1804, IF 1:1000, WB 1:1000) and α-tubulin (Sigma-Aldrich, DM1A, IF1:1000); goat polyclonal antibody against GFP, FITC-conjugated (Abcam, ab6662, IF 1:300).

Techniques: Staining, Control, Marker, Transfection, Two Tailed Test, Plasmid Preparation, Over Expression, Expressing

Journal: Nature Communications

Article Title: Cep295 is a conserved scaffold protein required for generation of a bona fide mother centriole

doi: 10.1038/ncomms12567

Figure Lengend Snippet: ( a ) 3D-SIM images showing centriolar distribution of Cep192 in control and Cep295-depleted HeLa cells (M: mother centriole; D: daughter centriole). Arrows point to the daughter centriole wall ( N =5). Dotted lines indicate the shape of centriole cylinders in this figure. Scale bar, 400 nm. ( b ) U2OS cells were transfected with Flag-tagged Cep192 plasmid for 24 h. The cells were fixed and stained with antibodies against Flag and Cep295 for STED microscope. The right panels show magnified views. Arrowhead points to the cap-like structure of Cep295 at the procentriole assembly site before recruitment of Cep192 ( N =10). Scale bar, 500 nm. The schematic illustrates the centriolar localization of Cep295 and Cep192. ( c ) STED images showing centriolar distribution of Cep295 in the presence or absence of HsSAS-6. HeLa cells were treated with control siRNA or HsSAS-6 siRNA for 48–60 h and stained with the indicated antibodies. To make sure the efficacy of HsSAS-6 depletion, we chose the Hs-SAS-6-depleted cells having only one mother centriole with a mono-polar spindle. Arrowhead shows centriolar distribution of Cep295 without the cartwheel structure ( N =3). Scale bar, 500 nm. ( d ) Control and Cep295-depleted HeLa cells were visualized by STED microscope using the indicated antibodies. Arrows point to poly-glutamylation of centriolar microtubules stained with GT335 antibody at the daughter centrioles ( N =3). Scale bar, 500 nm. ( e , f ) ( e ) HeLa cells treated with control siRNA or siRNA targeting endogenous Cep295 for 24 h were stained with antibodies against centrin1 (green) and Cep135 (red; a proximal component of centrioles). Nuclei are shown in blue. The arrow indicates defective recruitment of Cep135 to the disengaged daughter centriole. Scale bars, 5 μm in the low-magnified view, 1 μm in the inset. ( f ) Histograms represent frequency of late-mitotic cells with the indicated category in each condition. Values are mean percentages±s.e.m. from three independent experiments ( N =30 for each condition). ** P <0.01, (two-tailed t -test).

Article Snippet: The following primary antibodies were used in this study: Rabbit polyclonal antibodies against Cep295/KIAA1731 (Sigma, HPA038596, IF 1:1000, WB 1:1000), Cep192 (a gift from Laurence Pelletier, IF 1:1000), Cep192 (Bethyl laboratories, A302–324A, WB 1:1000), Cep152 (Bethyl laboratories, A302–480A, IF 1:1000), Cep152 (Bethyl laboratories, A302–479A, WB 1:1000), CP110 (a gift from Brian David Dynlacht, IF 1:500), CP110 (proteintech, 12780–1-AP, IF 1:500), CPAP (a gift from Pierre Gönczy, IF 1:500), CPAP/CENP-J (Proteintech, 11517-1-AP, IF 1:500), STIL (Abcam, ab89314, IF 1:500, WB 1:1000), Cep135 (Abcam, ab196809, IF 1:1000); mouse monoclonal antibodies against centrin-2 (Millipore, 20H5, IF 1:1000), HsSAS-6 (Santa Cruz Bio-technology, Inc., sc-81431, IF 1:500, WB 1:1000), Plk4 (Merck Millipore, clone 6H5, MABC544, IF 1:500), γ-tubulin (GTU88) (Sigma-Aldrich, T5192, IF 1:1000), Polyglutamylation Modification (GT335, mAb) (AdipoGen, AG-20B-0020-C100, IF 1:5000), FLAG-tag (Sigma, F1804, IF 1:1000, WB 1:1000) and α-tubulin (Sigma-Aldrich, DM1A, IF1:1000); goat polyclonal antibody against GFP, FITC-conjugated (Abcam, ab6662, IF 1:300).

Techniques: Control, Transfection, Plasmid Preparation, Staining, Microscopy, Two Tailed Test

Journal: Nature Communications

Article Title: Cep295 is a conserved scaffold protein required for generation of a bona fide mother centriole

doi: 10.1038/ncomms12567

Figure Lengend Snippet: ( a ) Physical interaction between endogenous Cep295 and Cep192. U2OS cells were transfected with Flag-tagged Cep192 full-length for 24 h. The proteins were then immunoprecipitated from cell lysates with Flag beads. Total cell lysate and immunoprecipitates (IPs) were analysed by western blotting using Flag, Cep295 or α-tubulin (loading control) antibodies. The same result was obtained in 293T cells. ( b ) Yeast two-hybrid assay showing the interaction between full-length Cep192 and full-length Cep295. The indicated clones were grown on the plates lacking histidine and containing 50 mM 3-AT at 30 °C. The same results were obtained from two independent clones for each combination. The outcome of the other clone is not shown. ( c ) Schematic of full-length Cep295 and the deletion mutants used for co-IP assays with Flag-tagged full-length Cep192 in U2OS cells. The sufficient and necessary regions for Cep192-binging in Cep295 are represented in pink and red, respectively. The DDC8-like domain, ALMS domain, and two evolutionarily conserved domains are indicated. ( d , e ) Co-immunoprecipitation assay in U2OS cells testing interaction between Flag-Cep192 and the indicated Cep295-GFP deletion mutants or between Cep295 fragment containing the Cep192-interacting region and N-terminal or C-terminal fragment of Cep192. The Flag-tagged proteins were immunoprecipitated using Flag beads from the cell lysate. Total cell lysates and IPs were analysed by western blotting using the indicated antibodies. ( f ) Three colour staining of Cep295-depleted HeLa cells expressing full-length Cep295 and the mutant protein lacking aa 1942–2431. The phenotype was analysed mostly in the first round of cell cycle (∼48 h after the RNAi treatment). The cells were analysed by TCS SP8 HSR system using antibodies against Cep192 (green), Cep295 (red) and CP110 (blue). Arrows point to the daughter centriole wall. M: mother centriole indicated as a cylinder. Scale bar, 1 μm. ( g ) For rescue experiments, Cep295-depleted HeLa cells were transfected with full-length Cep295 and the indicated mutant. Histograms represent frequency of interphase cells with the indicated number of γ-tubulin foci in each condition. Values are mean percentages±s.e.m from three independent experiments ( N >50 for each condition). * P <0.05 (two-tailed t -test).

Article Snippet: The following primary antibodies were used in this study: Rabbit polyclonal antibodies against Cep295/KIAA1731 (Sigma, HPA038596, IF 1:1000, WB 1:1000), Cep192 (a gift from Laurence Pelletier, IF 1:1000), Cep192 (Bethyl laboratories, A302–324A, WB 1:1000), Cep152 (Bethyl laboratories, A302–480A, IF 1:1000), Cep152 (Bethyl laboratories, A302–479A, WB 1:1000), CP110 (a gift from Brian David Dynlacht, IF 1:500), CP110 (proteintech, 12780–1-AP, IF 1:500), CPAP (a gift from Pierre Gönczy, IF 1:500), CPAP/CENP-J (Proteintech, 11517-1-AP, IF 1:500), STIL (Abcam, ab89314, IF 1:500, WB 1:1000), Cep135 (Abcam, ab196809, IF 1:1000); mouse monoclonal antibodies against centrin-2 (Millipore, 20H5, IF 1:1000), HsSAS-6 (Santa Cruz Bio-technology, Inc., sc-81431, IF 1:500, WB 1:1000), Plk4 (Merck Millipore, clone 6H5, MABC544, IF 1:500), γ-tubulin (GTU88) (Sigma-Aldrich, T5192, IF 1:1000), Polyglutamylation Modification (GT335, mAb) (AdipoGen, AG-20B-0020-C100, IF 1:5000), FLAG-tag (Sigma, F1804, IF 1:1000, WB 1:1000) and α-tubulin (Sigma-Aldrich, DM1A, IF1:1000); goat polyclonal antibody against GFP, FITC-conjugated (Abcam, ab6662, IF 1:300).

Techniques: Transfection, Immunoprecipitation, Western Blot, Control, Y2H Assay, Clone Assay, Co-Immunoprecipitation Assay, Staining, Expressing, Mutagenesis, Two Tailed Test

Journal: Nature Communications

Article Title: Cep295 is a conserved scaffold protein required for generation of a bona fide mother centriole

doi: 10.1038/ncomms12567

Figure Lengend Snippet: ( a ) Schematic of full-length Cep192 and the deletion mutants used for co-IP assays with endogenous Cep295 in U2OS cells. The minimal Cep295-binging region in Cep192 is represented in grey. ( b ) U2OS cells were transfected with Flag-Cep192 full-length and the indicated mutants. The cells were stained with antibodies against Cep192 (green) and CP110 (red). Scale bar, 1 μm. ( c , d ) U2OS cells expressing Flag-Cep192 full length or the deletion mutant proteins were immunoprecipitated with FLAG beads. Total cell lysates and IPs were analysed by western blotting using Cep295, Flag or tubulin antibodies. ( e ) GST pull-down assay showing the interaction between Cep295 and Cep192 fragments (aa 1727–2204 of Cep295 and aa 1501–2040 of Cep192) in vitro. These bacterially purified recombinant proteins contain the interacting regions that were identified by co-immunoprecipitation experiments. ( f , g ) For rescue experiments, Cep192-depleted U2OS cells were transfected with RNAi-resistant full-length Cep295 and the indicated mutant. Scale bar, 5 μm. Histograms represent frequency of bipolar mitotic spindles with the indicated number of γ-tubulin foci. Values are mean percentages±s.e.m from three independent experiments ( N =30 for each condition). * P <0.05 (two-tailed t -test). ( h , i ) Dominant negative effects of the Cep192 fragment that binds to Cep295. U2OS cells were transfected with full-length Cep295 and the indicated mutant. Scale bar, 1 μm. Histograms represent frequency of transfected cells with the indicated intensity of γ-tubulin foci. Values are mean percentages±s.e.m from three independent experiments ( N =30 for each condition). ** P <0.01 (two-tailed t -test).

Article Snippet: The following primary antibodies were used in this study: Rabbit polyclonal antibodies against Cep295/KIAA1731 (Sigma, HPA038596, IF 1:1000, WB 1:1000), Cep192 (a gift from Laurence Pelletier, IF 1:1000), Cep192 (Bethyl laboratories, A302–324A, WB 1:1000), Cep152 (Bethyl laboratories, A302–480A, IF 1:1000), Cep152 (Bethyl laboratories, A302–479A, WB 1:1000), CP110 (a gift from Brian David Dynlacht, IF 1:500), CP110 (proteintech, 12780–1-AP, IF 1:500), CPAP (a gift from Pierre Gönczy, IF 1:500), CPAP/CENP-J (Proteintech, 11517-1-AP, IF 1:500), STIL (Abcam, ab89314, IF 1:500, WB 1:1000), Cep135 (Abcam, ab196809, IF 1:1000); mouse monoclonal antibodies against centrin-2 (Millipore, 20H5, IF 1:1000), HsSAS-6 (Santa Cruz Bio-technology, Inc., sc-81431, IF 1:500, WB 1:1000), Plk4 (Merck Millipore, clone 6H5, MABC544, IF 1:500), γ-tubulin (GTU88) (Sigma-Aldrich, T5192, IF 1:1000), Polyglutamylation Modification (GT335, mAb) (AdipoGen, AG-20B-0020-C100, IF 1:5000), FLAG-tag (Sigma, F1804, IF 1:1000, WB 1:1000) and α-tubulin (Sigma-Aldrich, DM1A, IF1:1000); goat polyclonal antibody against GFP, FITC-conjugated (Abcam, ab6662, IF 1:300).

Techniques: Co-Immunoprecipitation Assay, Transfection, Staining, Expressing, Mutagenesis, Immunoprecipitation, Western Blot, Pull Down Assay, In Vitro, Purification, Recombinant, Two Tailed Test, Dominant Negative Mutation

Journal: Nature Communications

Article Title: Cep295 is a conserved scaffold protein required for generation of a bona fide mother centriole

doi: 10.1038/ncomms12567

Figure Lengend Snippet: A speculative model of the role of Cep295 in centriole biogenesis. Cep295 promotes recruitment of Cep192 onto the wall of a newly formed daughter centriole. Cep295 is also critical for the integrity of the proximal part of the daughter centriole and for stability of the resulting centriole in the next cell cycle. The events coordinated by Cep295 are thus critical for the ability of a new mother centriole to duplicate, recruit PCM components and act as the MTOC.

Article Snippet: The following primary antibodies were used in this study: Rabbit polyclonal antibodies against Cep295/KIAA1731 (Sigma, HPA038596, IF 1:1000, WB 1:1000), Cep192 (a gift from Laurence Pelletier, IF 1:1000), Cep192 (Bethyl laboratories, A302–324A, WB 1:1000), Cep152 (Bethyl laboratories, A302–480A, IF 1:1000), Cep152 (Bethyl laboratories, A302–479A, WB 1:1000), CP110 (a gift from Brian David Dynlacht, IF 1:500), CP110 (proteintech, 12780–1-AP, IF 1:500), CPAP (a gift from Pierre Gönczy, IF 1:500), CPAP/CENP-J (Proteintech, 11517-1-AP, IF 1:500), STIL (Abcam, ab89314, IF 1:500, WB 1:1000), Cep135 (Abcam, ab196809, IF 1:1000); mouse monoclonal antibodies against centrin-2 (Millipore, 20H5, IF 1:1000), HsSAS-6 (Santa Cruz Bio-technology, Inc., sc-81431, IF 1:500, WB 1:1000), Plk4 (Merck Millipore, clone 6H5, MABC544, IF 1:500), γ-tubulin (GTU88) (Sigma-Aldrich, T5192, IF 1:1000), Polyglutamylation Modification (GT335, mAb) (AdipoGen, AG-20B-0020-C100, IF 1:5000), FLAG-tag (Sigma, F1804, IF 1:1000, WB 1:1000) and α-tubulin (Sigma-Aldrich, DM1A, IF1:1000); goat polyclonal antibody against GFP, FITC-conjugated (Abcam, ab6662, IF 1:300).

Techniques:

Journal: Cell Discovery

Article Title: TRIP6 antagonizes the recruitment of A20 and CYLD to TRAF6 to promote the LPA2 receptor-mediated TRAF6 activation

doi: 10.1038/celldisc.2015.48

Figure Lengend Snippet: Depletion of TRIP6 eliminates LPA-induced NF-κB and MAP kinase activation in ovarian cancer cells. ( a ) TRIP6 associates with TRAF6 constitutively in SKOV-3 cells. SKOV-3 cells were starved for 24 h, followed by stimulation with LPA for 20 or 40 min. Endogenous TRAF6 was immunoprecipitated with anti-TRAF6 mouse monoclonal antibody or control mouse IgG, followed by immunoblotting with anti-TRIP6 rabbit antibody. The blot was reprobed with anti-TRAF6 rabbit antibody. The bottom panel shows the expression of endogenous TRIP6 in the whole-cell lysates. ( b ) Knockdown of TRIP6 attenuates LPA-induced IKK activation and IκBα phosphorylation. SKOV-3 cells stably expressing scrambled shRNA or TRIP6 shRNA were starved overnight, followed by stimulation with LPA for 30 or 60 min. Immunoblotting was performed to determine the levels of phospho-S32/36-IκBα, phospho-S176/180-IKKα/β, total IκBα, IKKβ, TRIP6 or TRAF6 in the whole-cell lysates. ( c ) Knockdown of TRIP6 reduces LPA-induced JNK activation. SKOV-3 cells stably expressing scrambled shRNA or TRIP6 shRNA were starved overnight, and then treated with LPA for 20, 40 or 80 min. Immunoblotting was performed to detect phosphorylated or total JNK, p38, TRIP6 or TRAF6 in the whole-cell lysates. ( d ) Depletion of TRIP6 by CRISPR/Cas9/sgRNA-mediated targeting of TRIP6 gene greatly eliminates LPA-stimulated IκBα phosphorylation and MAP kinase activation. SKOV-3 stable cell lines harboring Cas9 alone (sgControl) or Cas9/TRIP6 sgRNA (sgTRIP6-1, sgTRIP6-2) were treated with LPA for 30 or 60 min. Immunoblotting was performed to determine the levels of phosphorylated or total IκBα, IKKα/β, JNK, p38, ERK or TRIP6 in the whole-cell lysates. ( e ) Depletion of TRIP6 reduces LPA-promoted nuclear translocation of NF-κB p65. SKOV-3 cells harboring Cas9 alone (sgC) or Cas9/TRIP6 sgRNA (sgT-1, sgT-2) were starved overnight, followed by LPA stimulation for 90 min. Subcellular fractionation was performed in hypotonic buffer to separate the nucleus (pellet) from the cytosol (supernatant). The protein levels of NF-κB p65 or TRIP6 in each fraction were determined by immunoblotting. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and Histone H3 serve as cytosolic and nuclear markers, respectively. Data shown in ( a – e ) are representative of two to four independent experiments. ( f and g ) Depletion of TRIP6 reduces the basal and LPA-promoted NF-κB activity in SKOV-3 cells. SKOV-3 cells stably expressing shRNA (shScr, shTRIP6) ( f ) or Cas9/sgRNA (sgControl, sgTRIP6-1, sgTRIP6-2) ( g ) were transfected with the expression vectors of NF-κB-Luc and β-galactosidase. Cells were starved for 24 h, followed by LPA stimulation for 3 h. The NF-κB-driven luciferase activity was measured and normalized to the β-galactosidase activity. Data shown in ( f ) are the mean±s.e.m. of six independent experiments (* P <0.001 versus untreated shScr cells; ** P <0.001 versus treated shScr cells; Student’s t- test). Data shown in ( g ) are the mean±s.e.m. of four independent experiments (* P <0.001, ** P <0.05 versus untreated cells; *** P <0.001 versus treated sgControl cells; # P <0.001 versus untreated sgControl cells; Student’s t- test). ( h ) Knockdown of TRIP6 attenuates IL-6 activity in SKOV-3 cells. SKOV-3 cells stably expressing scrambled shRNA or TRIP6 shRNA were transfected with the expression vector of β-galactosidase and either pIL-6-Luc or pIL-6 mut-Luc with mutation in the NF-κB binding site. After stimulation for 3 h, the IL-6-driven luciferase activity was measured and normalized to the β-galactosidase activity. Data shown are the mean±s.e.m. of three independent experiments carried out in duplicates or triplicates (* P <0.05, ** P <0.01 versus untreated shScr cells; *** P <0.01 versus treated shScr cells; Student’s t- test). ( i ) Depletion of TRIP6 attenuates LPA-induced Bcl-xL expression. SKOV-3 cells stably expressing Cas9 alone (sgControl) or Cas9/TRIP6 sgRNA (sgTRIP6-1, sgTRIP6-2) were starved overnight, followed by LPA stimulation for 4 or 8 h. Immunoblotting was performed to detect the levels of Bcl-xL, TRIP6 or GAPDH. The result shown is representative of two independent experiments.

Article Snippet: The efficiency of TRIP6 knockout was determined by immunoblotting using anti-TRIP6 rabbit antibody (Bethyl Laboratories, Montgomery, TX, USA).

Techniques: Activation Assay, Immunoprecipitation, Control, Western Blot, Expressing, Knockdown, Phospho-proteomics, Stable Transfection, shRNA, CRISPR, Translocation Assay, Fractionation, Activity Assay, Transfection, Luciferase, Plasmid Preparation, Mutagenesis, Binding Assay

Journal: Cell Discovery

Article Title: TRIP6 antagonizes the recruitment of A20 and CYLD to TRAF6 to promote the LPA2 receptor-mediated TRAF6 activation

doi: 10.1038/celldisc.2015.48

Figure Lengend Snippet: TRIP6 recruits TRAF6 to the LPA2 receptor and promotes the LPA2 receptor-mediated JNK and NF-κB activation in a TRAF6-dependent manner. ( a ) Disruption of the LPA2 receptor binding to TRIP6 or knockdown of TRIP6 expression eliminates LPA-induced association of TRAF6 with the LPA2 receptor. The immortalized LPA1/2 DKO MEFs stably harboring an empty vector (mock), wild-type or C311A/C314A FLAG-LPA2 receptor, or FLAG-LPA2 receptor with mouse TRIP6 shRNA (shTRIP6) were starved for 5 h, followed by stimulation with 2 μ m LPA for 30 min. The FLAG-LPA2 receptor was immunoprecipitated with anti-FLAG M2 mouse monoclonal antibody-conjugated agarose beads, followed by immunoblotting with antibody specific to TRIP6, TRAF6 or FLAG epitope to determine the presence of endogenous TRIP6 or TRAF6 in the FLAG-LPA2 receptor complex. The bottom two panels show the expression of endogenous TRIP6 and TRAF6 in the whole-cell lysates. ( b ) Disruption of the LPA2 receptor binding to TRIP6 or knockdown of TRIP6 or TRAF6 attenuates LPA-induced IκBα phosphoylation and JNK activation. The immortalized LPA1/2 DKO MEFs stably harboring an empty vector (mock), wild-type or C311A/C314A FLAG-LPA2 receptor, or FLAG-LPA2 receptor with either mouse TRIP6 shRNA (shTRIP6) or mouse TRAF6 shRNA (shTRAF6) were starved for 5 h, followed by treatment with 2 μ m LPA for 30 min or 3 h. Immunoblotting was performed to determine the levels of phosphorylated or total IκBα, JNK, STAT3, TRIP6 or TRAF6 in the whole-cell lysates. ( c ) TRIP6 regulates the LPA2 receptor-mediated IκBα phosphorylation and JNK activation in a TRAF6-dependent manner. The LPA1/2 DKO MEFs stably expressing FLAG-LPA2 receptor with either scrambled shRNA or TRAF6 shRNA were transduced with lentivirus harboring either EGFP or EGFP-TRIP6. Cells were starved for 5 h, followed by LPA stimulation for 30 min. Immunoblotting was performed to determine the levels of phosphorylated or total IκBα, JNK, TRAF6, EGFP-TRIP6 or EGFP in the whole-cell lysates. Data shown in ( a – c ) are representative of three independent experiments. ( d – f ) Disruption of the LPA2 receptor binding to TRIP6 or knockdown of either TRIP6 or TRAF6 reduces the LPA2 receptor-mediated NF-κB and AP-1 activation. The LPA1/2-DKO MEF stable cell lines as indicated were transiently transfected with the expression vectors of β-galactosidase and either NF-κB-Luc ( d ), AP-1-Luc ( e ), IL-6-Luc or IL-6 mut-Luc with mutation in the NF-κB-binding site ( f ). After starvation for 5 h, cells were treated with LPA for another 3 h. Luciferase activity was measured and normalized to the β-galactosidase activity. In ( d ), data shown are the mean±s.e.m. of four independent experiments (* P <0.01, ** P <0.05 versus treated LPA2 MEFs; *** P <0.01, # P <0.001 versus untreated LPA2 MEFs; Student’s t-test ). In ( e ), data shown are the mean±s.e.m. of five independent experiments (* P <0.001, ** P <0.01 versus treated LPA2 MEFs; *** P <0.001, **** P <0.05, # P <0.01 versus untreated LPA2 MEFs; Student’s t-test ). In ( f ), data shown are the mean±s.e.m. of three independent experiments carried out in triplicates (* P <0.01 versus untreated LPA2 MEFs; ** P <0.05, *** P <0.01 versus treated LPA2 MEFs; Student’s t- test).

Article Snippet: The efficiency of TRIP6 knockout was determined by immunoblotting using anti-TRIP6 rabbit antibody (Bethyl Laboratories, Montgomery, TX, USA).

Techniques: Activation Assay, Disruption, Binding Assay, Knockdown, Expressing, Stable Transfection, Plasmid Preparation, shRNA, Immunoprecipitation, Western Blot, FLAG-tag, Phospho-proteomics, Transduction, Transfection, Mutagenesis, Luciferase, Activity Assay

Journal: Cell Discovery

Article Title: TRIP6 antagonizes the recruitment of A20 and CYLD to TRAF6 to promote the LPA2 receptor-mediated TRAF6 activation

doi: 10.1038/celldisc.2015.48

Figure Lengend Snippet: TRIP6 regulates LPA-induced K63-linked polyubiquitination of TRAF6, and antagonizes the recruitment of A20 and CYLD to TRAF6. ( a ) Knockdown of TRIP6 attenuates K63-linked polyubiquitination of transfected TRAF6. SKOV-3 cells stably expressing scrambled shRNA or TRIP6 shRNA were transfected with the expression vectors of HA-K63-ubiquitin and FLAG-TRAF6. Cells were starved for 6 h, followed by LPA stimulation for 1 h. Heat-denatured FLAG-TRAF6 was immunoprecipitated with anti-FLAG M2 monoclonal antibody-conjugated agarose beads, followed by immunoblotting using anti-HA antibody to detect K63-linked polyubiquitinated FLAG-TRAF6. After stripping, the immunoblot was reprobed with anti-FLAG antibody to detect immunoprecipitated FLAG-TRAF6. The bottom two panels show the expression of total HA-K63-ubiquitin and TRIP6 in the whole-cell lysates. ( b and c ) Depletion of TRIP6 eliminates LPA-stimulated K63-linked polyubiquitination of endogenous TRAF6. HEK293T cells stably harboring scrambled shRNA or TRIP6 shRNA ( b ), or SKOV-3 cells stably harboring Cas9 alone (sgC) or Cas9/TRIP6 sgRNA (sgTRIP6-1, sgTRIP6-2) ( c ) were transfected with the HA-K63-ubiquitin expression vector. Cells were starved overnight, followed by stimulation with LPA for 1 h. Heat-denatured endogenous TRAF6 was immunoprecipitated with anti-TRAF6 mouse monoclonal antibody, followed by immunoblotting with anti-HA antibody to detect K63-linked polyubiquitinated TRAF6. The immunoblot was reprobed with anti-TRAF6 rabbit polyclonal antibody to detect immunoprecipitated TRAF6. ( d ) TRIP6 barely enhances the in vitro autoubiquitination of TRAF6. The in vitro ubiquitination assay was performed by incubating E1, Ubc13/Uev1A (E2), ubiquitin and ATP without or with purified recombinant TRAF6 and TRIP6 at 30 °C for 1 h. After heat denaturation, TRAF6 was immunoprecipitated with anti-TRAF6 mouse monoclonal antibody, followed by immunoblotting with anti-ubiquitin rabbit antibody to detect autoubiquitinated TRAF6. The blot was reprobed with anti-TRAF6 rabbit antibody. The right panel shows coomassie blue staining of purified TRAF6 and TRIP6 used in this experiment. ( e ) LPA stimulation decreases the association of TRAF6 with CYLD, but promotes its binding to A20, whereas TRIP6 binds to TRAF6 constitutively and associates with NF-κB p65 following LPA stimulation. HEK293T cells expressing FLAG-TRAF6 were starved for 6 h, followed by LPA stimulation for the indicated times. FLAG-TRAF6 was immunoprecipitated with anti-FLAG M2 mouse monoclonal antibody-conjugated agarose beads, and co-immunoprecipitated endogenous CYLD, A20 or TRIP6 was detected by immunoblotting using rabbit antibody specific to each protein. Endogenous TRIP6 was immunoprecipitated with anti-TRIP6 mouse monoclonal antibody or control mouse IgG, followed by immunoblotting to detect co-immunoprecipitated endogenous NF-κB p65. The bottom four panels show the expression of endogenous CYLD, A20, TRIP6 or NF-κB p65 in the whole-cell lysates. ( f ) Overexpression of TRIP6 eliminates LPA-promoted interaction between TRAF6 and A20, and mildly attenuates the association of TRAF6 with CYLD in the absence of LPA. HEK293T cells expressing FLAG-TRAF6 alone or with HA-TRIP6 were starved for 6 h, followed by stimulation with LPA for 1.5 or 3 h. FLAG-TRAF6 was immunoprecipitated with anti-FLAG M2 mouse monoclonal antibody-conjugated agarose beads. Co-immunoprecipitated A20, CYLD or HA-TRIP6 was detected by immunoblotting using rabbit antibody specific to A20, CYLD or HA epitope. ( g ) TRIP6 physically interferes with the binding of TRAF6 to A20. HEK293T cells harboring an empty vector or FLAG-A20 were treated with LPA for 1.5 h, and then harvested in RIPA buffer. FLAG-A20 was immunoprecipitated with anti-FLAG M2 mouse monoclonal antibody-conjugated agarose beads. The beads were then aliquoted and incubated with 0.1 μg purified recombinant TRAF6 in the absence or presence of 0.1 μg purified recombinant TRIP6 at 4 °C for 3 h. After washing four times, the beads were subjected to immunoblotting with anti-TRAF6 rabbit antibody to detect co-immunoprecipitated recombinant TRAF6. The blot was stripped and reprobed with anti-FLAG antibody to detect immunoprecipitated FLAG-A20. The right panel shows coomassie blue staining of purified recombinant TRAF6 and TRIP6. ( h and i ) Depletion of TRIP6 greatly enhances the binding of TRAF6 to A20 and CYLD in SKOV-3 cells. SKOV-3 cell lines stably expressing shRNA (shScr, shTRIP6) ( h ) or Cas9/sgRNA (sgControl, sgTRIP6-1) ( i ) were starved overnight, followed by LPA treatment for various times as indicated. Endogenous TRAF6 was immunoprecipitated with anti-TRAF6 mouse monoclonal antibody or control mouse IgG, followed by immunoblotting with rabbit antibody specific to A20, CYLD, TRIP6 or TRAF6. The bottom four panels in each figure show the expression of endogenous A20, CYLD, TRIP6 or GAPDH in the whole-cell lysates. Data shown in each figure are representative of two to four independent experiments.

Article Snippet: The efficiency of TRIP6 knockout was determined by immunoblotting using anti-TRIP6 rabbit antibody (Bethyl Laboratories, Montgomery, TX, USA).

Techniques: Knockdown, Transfection, Stable Transfection, Expressing, shRNA, Ubiquitin Proteomics, Immunoprecipitation, Western Blot, Stripping Membranes, Plasmid Preparation, In Vitro, Purification, Recombinant, Staining, Binding Assay, Control, Over Expression, Incubation

Journal: Cell Discovery

Article Title: TRIP6 antagonizes the recruitment of A20 and CYLD to TRAF6 to promote the LPA2 receptor-mediated TRAF6 activation

doi: 10.1038/celldisc.2015.48

Figure Lengend Snippet: The TRAF6-binding motif and LIM domains 1 to 2 of TRIP6 are responsible for its interaction with the carboxyl-terminal domain and RING domain of TRAF6, respectively, and are both required for the blocking of A20 binding to TRAF6. ( a ) TRAF6 associates with the pre-LIM region and LIM domains of TRIP6. FLAG-TRIP6 or FLAG-TRIP6 mutant containing the pre-LIM region (residues 1–278) or three LIM domains (residues 220–476) was expressed in HEK 293T cells. FLAG proteins were immunoprecipitated with anti-FLAG M2 mouse monoclonal antibody-conjugated agarose beads, followed by immunoblotting with anti-TRAF6 antibody to detect co-immunoprecipitated endogenous TRAF6. The blot was reprobed with anti-FLAG antibody. The bottom panel shows the expression of endogenous TRAF6 in the whole-cell lysates. ( b ) The LIM domains of TRIP6 bind to the RING domain, but not zinc-finger domains, of TRAF6. FLAG-TRIP6 mutant containing the pre-LIM region (residues 1–278) or three LIM domains (residues 220–476) was co-expressed with an EGFP fusion protein containing either RING domain (residues 1–149) or zinc-finger (ZF) domains (residues 119–238) of TRAF6 in HEK293T cells. FLAG proteins were immunoprecipitated with anti-FLAG M2 mouse monoclonal antibody, followed by immunoblotting with anti-GFP antibody to detect co-immunoprecipitated EGFP fusion proteins. The blot was reprobed with anti-FLAG rabbit antibody. The bottom panel shows the expression of EGFP-tagged RING or zinc-finger domains of TRAF6 in the whole-cell lysates. ( c ) TRAF6 binds to LIM domains 1 to 2 of TRIP6 directly. Purified recombinant TRAF6 was incubated with GST, GST-TRIP6 or a GST-TRIP6 mutant containing either LIM domains 1 to 3 (residues 279–476), 2 to 3 (residues 340–476) or LIM3 domain (residues 398–476) at 4 °C for 3 h. TRAF6 pulled down by GST-TRIP6 or GST-TRIP6 mutant was detected by immunoblotting using anti-TRAF6 antibody. The bottom panel is an immunoblot of GST proteins detected with anti-GST antibody. ( d ) TRIP6 binds to the full-length TRAF6 or its carboxyl-terminal domain directly. Purified recombinant TRIP6 was incubated with GST, GST-TRAF6 or GST-CT mutant containing residues 239–530 of TRAF6 at 4 °C for 3 h. TRIP6 pulled down by GST-TRAF6 or GST-CT was detected by immunoblotting using anti-TRIP6 antibody. The bottom panel shows Ponceau S staining of GST fusion proteins. ( e ) The ability of TRIP6 to bind the carboxyl-terminal domain of TRAF6 is abolished by the E52A mutation. FLAG-TRIP6 or FLAG-E52A-TRIP6 was co-expressed with EGFP or EGFP-CT containing residues 239–530 of TRAF6 in HEK293T cells. Co-immunoprecipitation was performed as described in ( b ). Data shown in ( a – e ) are representative of two or three independent experiments. ( f ) Schematic structures of TRIP6 and TRAF6 and the domains responsible for their interactions. ( g ) The ability of TRIP6 to inhibit the association of TRAF6 with A20 requires both TRAF6-binding motif and LIM domains 1 to 2. HEK 293T cells transiently overexpressing FLAG-TRAF6 with either EGFP, EGFP-TRIP6, or one of the EGFP-TRIP6 deletion mutants were starved for 6 h, followed by addition of LPA for 90 min. Co-immunoprecipitation of FLAG-TRAF6 with endogenous A20 was performed as described in . The bottom two panels show the expression of EGFP, EGFP-tagged wild-type or mutant TRIP6, or A20 in the whole-cell lysates. ( h ) The ability of TRIP6 to block the recruitment of A20 to TRAF6 is reduced by the E52A mutation. HEK293T cells transiently expressing EGFP, EGFP-TRIP6 or EGFP-E52A-TRIP6 were starved for 6 h, and then treated with LPA for 90 min. Endogenous TRAF6 was immunoprecipitated with anti-TRAF6 mouse monoclonal antibody, followed by immunoblotting with anti-A20 or anti-GFP antibody to detect co-immunoprecipitated A20 or EGFP-TRIP6. The bottom three panels show the expression of A20, EGFP, EGFP-TRIP6 or EGFP-E52A-TRIP6 in the whole-cell lysates. Data shown in ( g ) and ( h ) are representative of three independent experiments.

Article Snippet: The efficiency of TRIP6 knockout was determined by immunoblotting using anti-TRIP6 rabbit antibody (Bethyl Laboratories, Montgomery, TX, USA).

Techniques: Binding Assay, Blocking Assay, Mutagenesis, Immunoprecipitation, Western Blot, Expressing, Purification, Recombinant, Incubation, Staining

Journal: Cell Discovery

Article Title: TRIP6 antagonizes the recruitment of A20 and CYLD to TRAF6 to promote the LPA2 receptor-mediated TRAF6 activation

doi: 10.1038/celldisc.2015.48

Figure Lengend Snippet: TRIP6 enhances TRAF6-NF-κB signaling by inhibiting the binding of A20 to TRAF6, but promotes LPA-induced TRAF6-JNK-AP-1 activation in an A20-independent manner. ( a ) The K63-linked polyubiquitination of TRAF6 is enhanced by wild-type TRIP6, but not E52A-TRIP6 mutant defective in blocking the recruitment of A20 to TRAF6. SKOV-3 cells stably expressing TRIP6 shRNA (SKOV-3-shTRIP6) were transiently transfected with the expression vectors of HA-K63-Ubiquitin, FLAG-TRAF6 and either EGFP, EGFP-TRIP6 or EGFP-E52A-TRIP6. After starvation for 24 h, cells were treated with LPA for 1 h. The levels of K63-linked polyubiquitinated FLAG-TRAF6 were determined as described in . The expression of HA-K63-ubiquitin, endogenous TRIP6, EGFP or EGFP-TRIP6 in the whole-cell lysates was detected by immunoblotting using antibody specific to HA, TRIP6 or GFP. ( b and c ) Mutation of E52 to Ala greatly eliminates the function of TRIP6 in promoting IκBα phosphorylation, but barely or only mildly attenuates its ability to promote LPA-induced JNK activation. SKOV-3 cells stably expressing TRIP6 shRNA ( b ) or Cas9/TRIP6 sgRNA (sgTRIP6-1) ( c ) were transiently transfected with EGFP- ( b ) or FLAG-tagged ( c ) wild-type or E52A TRIP6. Cells were starved for 24 h, followed by addition of LPA for 30 min. Immunoblotting was performed to detect the levels of indicated proteins in the whole-cell lysates. Data shown in ( a – c ) are representative of two or three independent experiments. ( d ) The effect of TRIP6 on promoting LPA-stimulated NF-κB activity is attenuated by the E52A mutation. SKOV-3-shTRIP6 cells were transiently transfected with pNF-κB-Luc, the β-galactosidase expression vector and either pEGFP, pEGFP-TRIP6 or pEGFP-E52A-TRIP6. Cells were starved for 24 h, followed by treatment with LPA for 3 h. The NF-κB-driven luciferase activity was determined and normalized to the β-galactosidase activity. Data shown are the mean±s.e.m. of four independent experiments done in duplicates (* P <0.001 versus untreated EGFP cells; ** P <0.01, *** P <0.05 versus treated EGFP cells; # P <0.05 versus untreated EGFP-TRIP6 cells; ## P <0.05 versus treated EGFP-TRIP6 cells; Student’s t-test ). ( e ) The effect of TRIP6 on promoting LPA-induced AP-1 activity is not significantly affected by the E52A mutation. The expression vectors of AP-1-Luc and β-galactosidase were co-transfected with either pEGFP, pEGFP-TRIP6 or pEGFP-E52A-TRIP6 into SKOV-3-shTRIP6 cells. After starvation for 24 h, cells were treated with LPA for 3 h. The AP-1-driven luciferase activity was determined and normalized to the β-galactosidase activity. Data shown are the mean±s.e.m. of three independent experiments done in triplicates (* P <0.01 versus untreated EGFP cells; ** P <0.001 versus treated EGFP cells; # P <0.05 versus untreated cells; Student’s t-test ). ( f ) A20 specifically inhibits LPA-stimulated IκBα phosphorylation, but not JNK activation; however, this effect is partially reversed by TRIP6 overexpression. SKOV-3 cells transiently overexpressing EGFP-A20 and/or FLAG-TRIP6 were starved in 0.1% fatty acid-free BSA-containing medium for 6 h, followed by stimulation with LPA for 30 min. Immunoblotting was performed to determine the levels of indicated proteins in the whole-cell lysates. Data shown are representative of two independent experiments. ( g ) The effect of A20 on the restriction of NF-κB activity is partially reversed by TRIP6 overexpression. NF-κB-Luc and β-galactosidase were transiently co-expressed with FLAG-A20 and/or FLAG-TRIP6 in SKOV-3 cells. Cells were starved overnight, followed by stimulation with LPA for 2 h. The NF-κB-driven luciferase activity was determined and normalized to the β-galactosidase activity. Data shown are the mean±s.e.m. of four independent experiments (* P <0.001, ** P <0.05 versus treated mock cells; *** P <0.05 versus untreated mock cells; # P <0.001, ## P <0.05 versus untreated cells; Student’s t-test ). The bottom three panels are representative immunoblots showing the expression of FLAG-TRIP6, FLAG-A20 or GAPDH in the whole-cell lysates.

Article Snippet: The efficiency of TRIP6 knockout was determined by immunoblotting using anti-TRIP6 rabbit antibody (Bethyl Laboratories, Montgomery, TX, USA).

Techniques: Binding Assay, Activation Assay, Mutagenesis, Blocking Assay, Stable Transfection, Expressing, shRNA, Transfection, Ubiquitin Proteomics, Western Blot, Phospho-proteomics, Activity Assay, Plasmid Preparation, Luciferase, Over Expression

Journal: Cell Discovery

Article Title: TRIP6 antagonizes the recruitment of A20 and CYLD to TRAF6 to promote the LPA2 receptor-mediated TRAF6 activation

doi: 10.1038/celldisc.2015.48

Figure Lengend Snippet: The function of TRIP6 in promoting the LPA2 receptor-mediated apoptotic resistance is in part mediated through the activated TRAF6-NF-κB signaling. ( a ) Knockdown of TRAF6 attenuates the LPA2 receptor-mediated protection from adriamycin-induced apoptosis. The LPA1/2 DKO MEFs stably expressing FLAG-LPA2 with scrambled shRNA or TRAF6 shRNA were pretreated with 10 μ m LPA for 1 h, followed by addition of 2 μ m adriamycin (Adr) for 8 h. Apoptosis was determined by caspase-3/7 activity assay (top panel). Data shown are the mean±s.e.m. of three independent experiments (* P <0.001 versus Adr treatment; ** P <0.01 versus LPA2-shScr MEFs treated with Adr and LPA; Student’s t-test ). Another set of cells was subjected to cell viability assay (bottom panel). After treatment, cells were incubated with 0.5 μ m calcein-AM and 0.1 μg ml −1 propidium iodide at room temperature for 10 min. Images of calcein-positive green fluorescent live cells and propidium iodide-positive dead cells were acquired under fluorescence microscope. Totally 500–1000 cells per sample were counted to determine the percentage of dead cells. Data shown are the mean±s.e.m. of three independent experiments (* P <0.001, ** P <0.01 versus LPA2-shScr MEFs treated with Adr; *** P <0.01 versus LPA2-shScr MEFs treated with Adr and LPA; Student’s t-test ). ( b ) Depletion of TRIP6 eliminates LPA-mediated protection from cisplatin-induced apoptosis in ovarian cancer cells. SKOV-3 stable cell lines expressing Cas9 (sgControl) or Cas9/TRIP6 sgRNA (sgTRIP6-1, sgTRIP6-2) were pretreated with 10 μ m LPA in 0.1% fatty acid-free BSA-containing medium for 1 h, followed by addition of 50 μ m cisplatin for 20 h. Apoptosis was determined by caspase-3/7 activity assay (top panel). Data shown are the mean±s.e.m. of three independent experiments (* P <0.01 versus sgControl cells treated with cisplatin; Student’s t-test ). The percentage of dead cells was determined by cell viability assay using calcein/propidium iodide double fluorescence staining as described above (bottom panel). Data shown are the mean±s.e.m. of three independent experiments (* P <0.05 versus sgControl cells treated with cisplatin; Student’s t-test ). ( c ) Adding TRIP6 back to the TRIP6-depleted SKOV-3 cells restores the antiapoptotic function of LPA; however, this effect is attenuated by the E52A mutation. SKOV-3 cells stably expressing Cas9/TRIP6 sgRNA (SKOV-3-sgTRIP6-1) were transiently transfected with an empty vector (mock), FLAG-TRIP6 or FLAG-E52A-TRIP6. Cells were pretreated with 10 μ m LPA for 1 h, followed by addition of 50 μ m cisplatin for 20 h. Apoptosis was determined by caspase-3/7 activity assay (top panel). Data shown are the mean±s.e.m. of three independent experiments (* P <0.01, ** P <0.05 versus cisplatin-treated cells; *** P <0.05 versus FLAG-TRIP6 cells treated with cisplatin and LPA; Student’s t-test ). The percentage of dead cells was determined by cell viability assay using calcein/propidium iodide double fluorescence staining as described above (bottom panel). Data shown are the mean±s.e.m. of three independent experiments (* P <0.05 versus FLAG-TRIP6 cells treated with cisplatin; ** P <0.05 versus FLAG-TRIP6 cells treated with cisplatin and LPA; Student’s t-test ). The right bottom panels are two representative immunoblots showing the expression of FLAG-TRIP6, FLAG-E52A-TRIP6 or GAPDH in the whole-cell lysates. ( d ) The effect of TRIP6 knockdown on the inhibition of LPA-mediated antiapoptotic function can be rescued by transiently overexpressing TRIP6; however, this effect is attenuated by the E52A mutation. SKOV-3 cells stably expressing TRIP6 shRNA were transiently transfected with an empty vector (mock), FLAG-TRIP6 or FLAG-E52A-TRIP6. Cells were treated with 10 μ m LPA, followed by addition of 50 μ m cisplatin for 20 h. Apoptosis was determined by caspase-3/7 activity assay. Data shown are the mean±s.e.m. of three independent experiments (* P <0.01, ** P <0.05 versus cisplatin-treated cells; *** P <0.05 versus FLAG-TRIP6 cells treated with cisplatin and LPA; Student’s t-test ). The bottom two panels are representative immunoblots showing the expression of endogenous TRIP6, FLAG-TRIP6, FLAG-E52A, or GAPDH in the whole-cell lysates.

Article Snippet: The efficiency of TRIP6 knockout was determined by immunoblotting using anti-TRIP6 rabbit antibody (Bethyl Laboratories, Montgomery, TX, USA).

Techniques: Knockdown, Stable Transfection, Expressing, shRNA, Activity Assay, Viability Assay, Incubation, Fluorescence, Microscopy, Staining, Mutagenesis, Transfection, Plasmid Preparation, Western Blot, Inhibition

Journal: Cell Discovery

Article Title: TRIP6 antagonizes the recruitment of A20 and CYLD to TRAF6 to promote the LPA2 receptor-mediated TRAF6 activation

doi: 10.1038/celldisc.2015.48

Figure Lengend Snippet: TRAF6 regulates the functions of TRIP6 in NF-κB p65 binding, c-Src-dependent tyrosine phosphorylation of TRIP6 and LPA2 receptor-mediated cell migration. ( a ) Knockdown of TRAF6 eliminates LPA-induced association of TRIP6 with NF-κB p65 in the cytosol, and reduces the levels of nuclear NF-κB p65. LPA1/2 DKO MEFs stably expressing LPA2 receptor and either scrambled shRNA or TRAF6 shRNA were starved for 6 h, followed by LPA stimulation for 1 h. Cells were subjected to subcellular fractionation to separate nuclei from the cytosol. Immunoblotting was performed to detect the expression of NF-κB p65, TRIP6 and TRAF6 in each fraction (bottom panel). Histone H3 and GAPDH serve as nuclear and cytosolic markers, respectively. The cytosolic TRIP6 was further immunoprecipitated with anti-TRIP6 mouse monoclonal antibody or control mouse IgG, followed by immunoblotting using anti-NF-κB p65 rabbit antibody to detect the association of TRIP6 with NF-κB p65 in the cytosol (top panel). ( b ) Overexpression of ligase-defective C70A-TRAF6 eliminates the association of TRIP6 with NF-κB p65. HEK293T cells transiently expressing FLAG-TRIP6, HA-K63-ubiquitin and either EGFP, EGFP-TRAF6 or EGFP-C70A-TRAF6 were starved for 6 h, followed by LPA stimulation for 1 h. Subcellular fractionation was performed in hypotonic buffer to separate nuclei (pellet) from the cytosol (supernatant). FLAG-TRIP6 in each fraction was immunoprecipitated with anti-FLAG M2 mouse monoclonal antibody-conjugated agarose beads, followed by immunoblotting to detect co-immunoprecipitated endogenous NF-κB p65. The immunoblot was reprobed with anti-FLAG antibody to detect precipitated FLAG-TRIP6. The expression of HA-ubiquitin or EGFP fusion proteins in each fraction was detected by immunoblotting using anti-HA or anti-GFP antibody. GAPDH and Histone H3 serve as cytosolic and nuclear markers, respectively. ( c and d ) Knockdown of TRAF6 attenuates c-Src kinase activity and reduces LPA-stimulated tyrosine phosphorylation of TRIP6. SYF+c-Src MEFs ( c ) or LPA1/2 DKO+LPA2 MEFs ( d ) were infected with lentivirus harboring scrambled shRNA or mouse TRAF6-specific shRNA. Cells were starved for 8 h, followed by LPA stimulation for the indicated times. Endogenous TRIP6 was immunoprecipitated with anti-TRIP6 mouse monoclonal antibody, followed by immunoblotting using HRP-conjugated anti-phospho-tyrosine antibody and anti-TRIP6 rabbit antibody, respectively. The bottom panels show the expression of pY416-c-Src, total c-Src, TRAF6 or TRIP6 in the whole-cell lysates. Data shown in ( a – d ) are representative of two to four independent experiments. ( e ) Knockdown of TRAF6 reduces LPA-induced cell migration and attenuates the function of TRIP6 in promoting the LPA2 receptor-mediated cell migration. The LPA1/2 DKO MEFs stably expressing FLAG-LPA2 receptor and either scramble shRNA or TRAF6 shRNA were transduced with lentivirus harboring EGFP or EGFP-TRIP6. LPA was added to the lower chamber of transwells, and cells were allowed to migrate for 6 h. Cells that migrated to the bottom filters of the transwells were fixed and stained with crystal violet. The relative migration rate was defined as the fold-increase of migrated cells compared to untreated shScr+EGFP cells. Data shown are the mean±s.e.m. of three independent experiments (* P <0.05 versus treated shScr+EGFP cells; ** P <0.05 versus untreated shScr+EGFP cells; # P <0.05 versus untreated cells; Student’s t-test ).

Article Snippet: The efficiency of TRIP6 knockout was determined by immunoblotting using anti-TRIP6 rabbit antibody (Bethyl Laboratories, Montgomery, TX, USA).

Techniques: Binding Assay, Phospho-proteomics, Migration, Knockdown, Stable Transfection, Expressing, shRNA, Fractionation, Western Blot, Immunoprecipitation, Control, Over Expression, Ubiquitin Proteomics, Activity Assay, Infection, Transduction, Staining

Journal: Cell Discovery

Article Title: TRIP6 antagonizes the recruitment of A20 and CYLD to TRAF6 to promote the LPA2 receptor-mediated TRAF6 activation

doi: 10.1038/celldisc.2015.48

Figure Lengend Snippet: A model for the cooperative regulation between TRIP6 and TRAF6 in the LPA2 receptor signaling. Upon LPA stimulation, TRIP6 is targeted to the plasma membrane, where it forms a complex with the LPA2 receptor and NHERF2, and serves as a scaffold to recruit TRAF6, c-Src and AKT. Together, they coordinate to activate ERK, JNK, p38, AKT and induce c-Src-dependent tyrosine phosphorylation of TRIP6. Through direct binding to TRAF6, TRIP6 antagonizes the recruitment of A20 and CYLD to TRAF6, thus sustaining the E3 ligase activity of TRAF6 upon LPA stimulation. This promotes the IKK-dependent phosphorylation and degradation of IκB, and allows TRIP6 to bind NF-κB p65. Once translocated to the nucleus, TRIP6 can further serve as a coactivator of NF-κB p65 and AP-1 to activate their target genes involved in apoptotic resistance, chronic inflammation, proliferation and invasion.

Article Snippet: The efficiency of TRIP6 knockout was determined by immunoblotting using anti-TRIP6 rabbit antibody (Bethyl Laboratories, Montgomery, TX, USA).

Techniques: Clinical Proteomics, Membrane, Phospho-proteomics, Binding Assay, Activity Assay

Journal: Scientific reports

Article Title: Tracing the invisible mutant ADNP protein in Helsmoortel-Van der Aa syndrome patients.

doi: 10.1038/s41598-024-65608-x

Figure Lengend Snippet: Figure 1. Common commercially available ADNP antibodies give rise to non-specific binding. HEK293T, HeLa, SHSY-5Y and a lymphoblastoid control cell line (LCL) were lysed in RIPA buffer and used as protein samples for the assessment of the published ADNP antibodies. Samples were blocked and incubated in 5% blocking-grade non-fat dry milk/TBST with the optimized dilution listed in Table 3. The predicted molecular weight of ADNP is 124 kDa. However, only non-specific signals were detectable. GAPDH was used as a loading control. The datasheet of the tested antibodies indicated that whole or nuclear extracts from HeLa cells should be used as a positive control, which fails to raise a reliable ADNP signal in all tested antibody conditions.

Article Snippet: Recently, Aviva Systems developed an N-terminal antibody raised against the amino acids 35–82 of a human recombinant ADNP peptide (ARP39186_P050) with a predicted reactivity in human, mouse, and rat samples.

Techniques: Binding Assay, Control, Incubation, Blocking Assay, Molecular Weight, Positive Control

Journal: Scientific reports

Article Title: Tracing the invisible mutant ADNP protein in Helsmoortel-Van der Aa syndrome patients.

doi: 10.1038/s41598-024-65608-x

Figure Lengend Snippet: Figure 2. Verification of the specificity of an N-terminal ADNP antibody (Aviva Systems) by performing a blocking peptide competition assay. (A) HEK293T, HeLa, SHSY-5Y and a control lymphoblastoid cell line (LCL) were lysed in RIPA buffer and used as protein samples for the assessment of N-terminal antibody of Aviva systems in a 1:1000 dilution. GAPDH was used as a loading control. The predicted molecular weight of ADNP is 124 kDa. The antibody recognizes ADNP specifically at 150 kDa in HEK293T, HeLa and SHSY-5Y cell lines, but a faint signal ranging from 75 to 150 kDa in the control LCL. (B) Western blot analysis of the blocking peptide competition assay. Supplementation of the immunization peptide in a 5 × excess to antibody concentration reduced the signal detected at 75- 150 kDa in all tested cell lines. Non-specific binding was detected after use of the immunization peptide presenting as a faint signal below the 37 kDa marker.

Article Snippet: Recently, Aviva Systems developed an N-terminal antibody raised against the amino acids 35–82 of a human recombinant ADNP peptide (ARP39186_P050) with a predicted reactivity in human, mouse, and rat samples.

Techniques: Blocking Assay, Competitive Binding Assay, Control, Molecular Weight, Western Blot, Concentration Assay, Binding Assay, Marker

Journal: Scientific reports

Article Title: Tracing the invisible mutant ADNP protein in Helsmoortel-Van der Aa syndrome patients.

doi: 10.1038/s41598-024-65608-x

Figure Lengend Snippet: Figure 3. A polyclonal N-terminal ADNP antibody from Aviva Systems detects ADNP specifically in murine and rat tissues and suggests proteolytic processing of the protein in the human brain. Cerebellum, frontal cortex or lobe, hippocampus and whole brains of control mice, rats and humans were lysed in RIPA buffer and used as protein samples for the assessment of N-terminal antibody of Aviva systems. (A–C) The predicted molecular weight of ADNP is 124 kDa. The antibody recognizes ADNP in a range of 145 kDa with (E) additional lower mass signal of 85 kDa in all human brain regions. (B–D–F) Western blot analysis of the blocking peptide competition assay. Supplementation of the immunization peptide in a 5 × excess to antibody concentration reduced the signal observed at 145 kDa in all tested cell lines. Importantly, the 85 kDa band suggestive for proteolytic cleavage as well as degraded ADNP signal disappeared completely after immunization peptide supplementation. GAPDH was used as a loading control.

Article Snippet: Recently, Aviva Systems developed an N-terminal antibody raised against the amino acids 35–82 of a human recombinant ADNP peptide (ARP39186_P050) with a predicted reactivity in human, mouse, and rat samples.

Techniques: Control, Molecular Weight, Western Blot, Blocking Assay, Competitive Binding Assay, Concentration Assay

Journal: Scientific reports

Article Title: Tracing the invisible mutant ADNP protein in Helsmoortel-Van der Aa syndrome patients.

doi: 10.1038/s41598-024-65608-x

Figure Lengend Snippet: Figure 4. Three independent commercially available C-terminal polyclonal ADNP antibodies detect ADNP specifically in different in vitro sample materials and show clear instability of the protein. HEK293T, HeLa, SHSY-5Y and a lymphoblastoid cell line (LCL) were lysed in RIPA buffer and used as protein samples for three different C-terminal ADNP antibodies. GAPDH was used as a loading control. The predicted molecular weight of ADNP is 124 kDa. All the tested antibodies recognized ADNP with a molecular weight of 150 kDa. Samples were blocked and incubated in 5% blocking-grade non-fat dry milk/TBST with the optimized dilution listed in Table 3.

Article Snippet: Recently, Aviva Systems developed an N-terminal antibody raised against the amino acids 35–82 of a human recombinant ADNP peptide (ARP39186_P050) with a predicted reactivity in human, mouse, and rat samples.

Techniques: In Vitro, Control, Molecular Weight, Incubation, Blocking Assay

Journal: Scientific reports

Article Title: Tracing the invisible mutant ADNP protein in Helsmoortel-Van der Aa syndrome patients.

doi: 10.1038/s41598-024-65608-x

Figure Lengend Snippet: Figure 5. Different C-terminal ADNP antibodies detect ADNP in the range of 150 kDa and suggest proteolytic processing of the protein in the brain. Cerebellum, frontal cortex or lobe, hippocampus and whole brains of control mice, rats, and humans were lysed in RIPA buffer and used as protein samples for the assessment with three C-terminal antibodies with the optimized dilutions listed in Table 3. GAPDH was used as a loading control. The predicted molecular weight of ADNP is 124 kDa. (A)C) Murine samples indicate detection of ADNP in the range of 150 kDa with bands suggesting proteolytic processing at 50 kDa. (D–F) Rat samples indicate detection of ADNP in the range of 150 kDa with bands indicating proteolytic processing at 82 kDa after incubation with the C-terminal Abcam antibody. (G–I) Human brain samples indicate detection of ADNP at different molecular weights of 124 – 150 kDa in the adult frontal lobe and hippocampus and highlight the antibody differences in detection of ADNP. The three tested antibodies showed strong band signals at lower molecular weights, which could indicate proteolytic cleavage or degradation of the protein.

Article Snippet: Recently, Aviva Systems developed an N-terminal antibody raised against the amino acids 35–82 of a human recombinant ADNP peptide (ARP39186_P050) with a predicted reactivity in human, mouse, and rat samples.

Techniques: Control, Molecular Weight, Incubation

Journal: Scientific reports

Article Title: Tracing the invisible mutant ADNP protein in Helsmoortel-Van der Aa syndrome patients.

doi: 10.1038/s41598-024-65608-x

Figure Lengend Snippet: Figure 6. Unambiguous detection of ADNP using homozygous CRISPR/Cas9 endonuclease-mediated Adnp knockout cell lines. mESCs containing either wild-type, homozygous mutants, or complete Adnp knockout were lysed in RIPA buffer and used as protein samples for the assessment with an N-terminal ADNP, 3x-DYKDDDDK, and C-terminal ADNP antibodies with the optimized dilutions listed in Table 1. GAPDH was used as a loading control. The predicted molecular weight of ADNP is 124 kDa. (A) The N-terminal antibody (Aviva Systems) recognizes ADNP in a range above its observed 150 kDa molecular weight with additional lower mass signal of 37—65 kDa in Adnp homozygous and parental control mESCs. (B) Supplementation of the immunization peptide in a 5 × excess to antibody concentration reduced all signals observed mESC lines, indicating that the N-terminal antibody does not bind ADNP specifically in mESCs. (C) Detection of wild- type and homozygous Adnp mutants by means of a C-terminal 3x-DYKDDDDK (Flag) epitope tag. Wild-type ADNP was detected in at 150 kDa in the C-terminal 3x-DYKDDDDK CRISPR/Cas9 engineered mESC line using a DYKDDDDK antibody. Truncated ADNP mutants, p.Tyr718* and p.Lys407Valfs*31, were detected at a lower molecular weight of 80 kDa, respectively 48 kDa. (D–F) Wild-type ADNP detection by means of three different C-terminal antibodies in mESC lines. Wild-type ADNP was detected with a strong signal at 150 kDa in the parental control line with a rather decreased signal in the C-terminal 3x-DYKDDDDK CRISPR/Cas9 engineered mESC line. Disappearance of the 150 kDa band was observed in the mESC line with complete Adnp homozygosity, indicating a reliable molecular weight of 150 kDa for ADNP.

Article Snippet: Recently, Aviva Systems developed an N-terminal antibody raised against the amino acids 35–82 of a human recombinant ADNP peptide (ARP39186_P050) with a predicted reactivity in human, mouse, and rat samples.

Techniques: CRISPR, Knock-Out, Control, Molecular Weight, Concentration Assay, FLAG-tag

Journal: Scientific reports

Article Title: Tracing the invisible mutant ADNP protein in Helsmoortel-Van der Aa syndrome patients.

doi: 10.1038/s41598-024-65608-x

Figure Lengend Snippet: Figure 7. Unambiguous detection of ADNP using an N-terminal GFPSpark and N-DYKDDDDK (Flag) tag expression vector. (A) Western blot analysis of HEK293T cell lysates overexpressing wild-type ADNP- GFPSpark and mutated constructs using an anti-GFP antibody. (B) Western blot analysis of HEK293T cell lysates overexpressing wild-type ADNP-GFPSpark and mutated constructs using the N-terminal ADNP antibody (Aviva Systems). (C) Western blot analysis of HEK293T cell lysates overexpressing wild-type ADNP- DYKDDDDK (Flag) and mutated constructs using an anti-DYKDDDDK antibody. (D) Western blot analysis of HEK293T cell lysates overexpressing wild-type ADNP-DYKDDDDK and mutant constructs using the N-terminal ADNP antibody (Aviva Systems). The observed molecular weight of wild-type ADNP-GFPSpark is 175 kDa (including 25 kDa GFPSpark tag), respectively ADNP-DYKDDDDK 150 kDa, with each of their mutants showing a lower molecular weight as a consequence of the truncating mutations. Detection with antibodies for GFP, DYKDDDDK (Flag), and ADNP gave comparable results. GAPDH was used as a loading control in all experiments.

Article Snippet: Recently, Aviva Systems developed an N-terminal antibody raised against the amino acids 35–82 of a human recombinant ADNP peptide (ARP39186_P050) with a predicted reactivity in human, mouse, and rat samples.

Techniques: FLAG-tag, Expressing, Plasmid Preparation, Western Blot, Construct, Mutagenesis, Molecular Weight, Control

Journal: Scientific reports

Article Title: Tracing the invisible mutant ADNP protein in Helsmoortel-Van der Aa syndrome patients.

doi: 10.1038/s41598-024-65608-x

Figure Lengend Snippet: Figure 8. Western blotting of ADNP in a HCT116 colon cancer cell line, carrying the prevalent heterozygous p.Tyr719* mutation. HCT116 cells containing a wild-type and p.Tyr719* mutant allele were lysed in RIPA buffer and used as protein samples for the assessment with an N-terminal antibody, 3x-DYKDDDDK, HA-tag, and C-terminal ADNP antibodies with the optimized dilutions listed in Table 1. GAPDH was used as a loading control in all experiment. The predicted molecular weight of ADNP is 124 kDa. (A) The N-terminal antibody (Aviva Systems) recognizes ADNP in a range above its observed 150 kDa molecular weight an additional signal of 45 kDa, indicating proteolytic cleavage or non-specific binding. (B) Administration of the immunization peptide in a 5 × excess to antibody concentration reduced all signals, indicating that the N-terminal antibody does not bind ADNP specifically in HCT116 cells. (C) Detection of wild-type ADNP by means of the 3x-DYKDDDDK (Flag) epitope tag. Wild-type ADNP was detected in at 182 kDa in the 3xFlag-V5-loxP- neonGreen/3xHA-loxP-mCherry engineered line using a DYKDDDDK antibody, 32 kDa by tag insertion. (D) Detection of mutant ADNP by means of the HA-epitope tag. A truncated mutant p.Tyr719 ADNP protein was detected in at 105 kDa in the 3xFlag-V5-loxP-neonGreen/3xHA-loxP-mCherry engineered line using a HA-antibody, 25 kDa above its predicted molecular weight by tag insertion. Instability of the truncated protein was observed by a degrading smear. (E–G) Wild-type ADNP detection by means of three different C-terminal antibodies. Non-processed ADNP was detected with a strong signal at 150 kDa in the control line and at a molecular weight of 182 kDa in the genome-edited cell line. In both cases, a degrading smear was observed, indicating instability of the wild-type protein.

Article Snippet: Recently, Aviva Systems developed an N-terminal antibody raised against the amino acids 35–82 of a human recombinant ADNP peptide (ARP39186_P050) with a predicted reactivity in human, mouse, and rat samples.

Techniques: Western Blot, Mutagenesis, Control, Molecular Weight, Binding Assay, Concentration Assay, FLAG-tag

Journal: Scientific reports

Article Title: Tracing the invisible mutant ADNP protein in Helsmoortel-Van der Aa syndrome patients.

doi: 10.1038/s41598-024-65608-x

Figure Lengend Snippet: Figure 9. Western blotting of ADNP in human induced pluripotent stem cells (hiPSCs), carrying distinct heterozygous ADNP mutations mediated by CRIPSR/Cas9. (A, B) hiPSCs were lysed in RIPA buffer and analyzed by western blotting with the N-terminal antibody (Aviva Systems) with application of our blocking peptide competition assay. Here, no reliable ADNP signal was detected. The molecular weight of the ADNP mutant lines is expected to decrease to 127 kDa for the Asn832Lysfs*81, respectively to 48 kDa for the lys408Valfs*31 line. However, no signal is observed at the predicted weight for the mutations. (C–E) The C-terminal antibodies of Protein Technology, Abcam, and the Sarma Laboratory were able to visualize wild- type ADNP at 150 kDa. Possessing the desired epitope for mutant ADNP detection, the C-terminal antibody of Protein technology was not able to capture the predicted truncated protein. GAPDH was used as a loading control. (F) The ADNP signal was quantified determining the ratio of the wild-type protein in mutant to control cell lines. Here, the relative ADNP expression decreased in the Asn832Lysfs*81 cell line compared to the control, whereas mutant-to-wild-type expression ratio showed a higher signal with the antibodies of Protein Technology and Abcam in the lys408Valfs*31 cell line.

Article Snippet: Recently, Aviva Systems developed an N-terminal antibody raised against the amino acids 35–82 of a human recombinant ADNP peptide (ARP39186_P050) with a predicted reactivity in human, mouse, and rat samples.

Techniques: Western Blot, Blocking Assay, Competitive Binding Assay, Molecular Weight, Mutagenesis, Control, Expressing

Journal: Scientific reports

Article Title: Tracing the invisible mutant ADNP protein in Helsmoortel-Van der Aa syndrome patients.

doi: 10.1038/s41598-024-65608-x

Figure Lengend Snippet: Figure 10. Absence of a mutant ADNP protein after immunoblotting of different lymphoblastoid cell lines from Helsmoortel-Van der Aa syndrome patients. (A) LCLs of four control subjects and six patients were lysed in RIPA buffer and analyzed by western blotting with the N-terminal antibody (Aviva Systems). The expected wild-type ADNP signal presented at 150 kDa together with two non-specific bands at 50 kDa and 75 kDa with no difference in expression (p = 0.42; ns) of the wild-type protein. However, the ADNP mutants at a lower molecular weight of 127 kDa for the Asn832Lysfs*81 and Leu831Ilefs*82 mutations, respectively to 45 kDa for the Ser404* mutation, and to 10 kDa for the cell line carrying the Gln40* mutation could not be visualized. (B) Administration of the immunization peptide in a 5 × excess to antibody concentration reduced all signals, indicating that the N-terminal antibody recognized ADNP specifically in LCLs alongside non-specific band signals. (C-E) C-terminal antibodies detected wild-type ADNP at a molecular weight of 150 kDa. No mutant ADNP was observed with the antibody of Protein Technology which is capable to recognize a part of the truncated Asn832Lysfs*81 and Leu831Ilefs*82 mutations. (F) All C-terminal antibodies visualized wild-type ADNP at 150 kDa, with only the Abcam (p = 0.04; *) and Sarma Laboratory (p = 0.02; *) antibodies showing the expected reduction of ADNP in LCLs of Helsmoortel-Van der Aa syndrome patients. GAPDH was used as a loading control.

Article Snippet: Recently, Aviva Systems developed an N-terminal antibody raised against the amino acids 35–82 of a human recombinant ADNP peptide (ARP39186_P050) with a predicted reactivity in human, mouse, and rat samples.

Techniques: Mutagenesis, Western Blot, Control, Expressing, Molecular Weight, Concentration Assay

Journal: Scientific reports

Article Title: Tracing the invisible mutant ADNP protein in Helsmoortel-Van der Aa syndrome patients.

doi: 10.1038/s41598-024-65608-x

Figure Lengend Snippet: Figure 11. Wild-type and mutant ADNP enrichment through immunoprecipitation. The N-terminal sc-F5 ADNP IP-competent antibody was crosslinked to agarose beads and sequentially eluted in fractions (input; flow-through; three consecutive washes, W1-W3; and the immunoprecipitated fracted. IgG non-reactive beads were used as a negative control. In each lane, 20 μg of protein was separated by SDS-PAGE electrophoresis. GAPDH has been used as loading control for all western blots, and critical assessment of the accuracy of the IP method. (A) Immunoprecipitation assay of recombinant wild-type (WT) ADNP and truncating mutants (p.Tyr719*; p.Arg730*; p.Asn832Lysfs*81) in HEK293T overexpression lysates. (B) Immunoprecipitation assay of native wild-type (WT) ADNP and truncating mutants in protein extracts of LCLs derived from a control subject (CTR) and patients with the p.Ser404*, p.Leu831Ilefs*82, or p.Asn832Lysfs*81 ADNP mutation.

Article Snippet: Recently, Aviva Systems developed an N-terminal antibody raised against the amino acids 35–82 of a human recombinant ADNP peptide (ARP39186_P050) with a predicted reactivity in human, mouse, and rat samples.

Techniques: Mutagenesis, Immunoprecipitation, Negative Control, SDS Page, Electrophoresis, Control, Western Blot, Recombinant, Over Expression, Derivative Assay

Journal: PLoS Pathogens

Article Title: Interleukin 16 contributes to gammaherpesvirus pathogenesis by inhibiting viral reactivation

doi: 10.1371/journal.ppat.1008701

Figure Lengend Snippet: (A) 1×10 6 SL-1 and SL-3 cells were cultured for 24 hr, whole-cell lysate (WCL) and supernatant (SUP) were prepared and subjected to cytokine array analyses. Each cytokine has one pair of duplicate spots. A1-2, A23-24, and F1-2 are experimental positive control, and F23-24 is an experimental negative control. (B) C57BL/6 mice were intranasally inoculated with 5×10 4 PFU of MHV68-H2bYFP or mock inoculated with PBS. At day 16 post-infection, splenocytes were isolated and subjected to flow cytometry, the flow plot represented the strategy gating YFP+ MHV68 infected cells (left panel); serum was prepared from 10 virus-infected mice or mock-infected mice, followed by IL16 ELISA assay (right panel). Histograms represented mean ±SD of 10 individual mice (two experiments, n = 5 for each experiment). p value was determined by two-tailed unpaired t-test. (C) WT MEFs were infected with MHV68 at an MOI of 1, total RNA was isolated from infected cells harvested at the indicated time points and subjected to qRT-PCR analyses with specific primers corresponding to IL16 and MHV68 ORF50 gene. The relative RNA amount was normalized to GAPDH in each sample. Histograms represented the mean of three independent biological replicates ±SD, p value was determined by two-tailed unpaired t-test, p ≤ 0.05 represents significance.

Article Snippet: Mouse IL16 Antibody (842373) was from R&D Systems.

Techniques: Cell Culture, Positive Control, Negative Control, Infection, Isolation, Flow Cytometry, Virus, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Quantitative RT-PCR

Journal: PLoS Pathogens

Article Title: Interleukin 16 contributes to gammaherpesvirus pathogenesis by inhibiting viral reactivation

doi: 10.1371/journal.ppat.1008701

Figure Lengend Snippet: (A) Intracellular staining of IL16 in splenocytes isolated from IL16+/+, IL16+/-, and IL16-/- mice. (B) Immunoblot detection of IL16 expression in splenocytes isolated from IL16+/+, IL16+/-, and IL16-/- mice. (C) Representative flow plots showed flow cytometric analyses of splenocytes from IL16+/+ (WT) and IL16-/- (KO) mice. (D) The statistic analyses of CD4+ T, CD8+ T, and B cells (Left panel); follicular (FC) B, marginal zone (MZ) B, and mature B cells (Right panel) in splenocytes from WT and IL16 KO mice. Histograms represented mean ±SD of 8 individual mice (two experiments, n = 4 for each experiment). ns = not significant.

Article Snippet: Mouse IL16 Antibody (842373) was from R&D Systems.

Techniques: Staining, Isolation, Western Blot, Expressing

Journal: PLoS Pathogens

Article Title: Interleukin 16 contributes to gammaherpesvirus pathogenesis by inhibiting viral reactivation

doi: 10.1371/journal.ppat.1008701

Figure Lengend Snippet: (A) WT and IL16 KO MEFs were infected with MHV68 at an MOI of 1 or 0.05. The infected cells were harvested at the indicated time points and immunoblot analyses were performed with specific antibodies as indicated. Actin was used as a loading control. (B) WT and IL16 KO MEFs were infected with MHV68 at an MOI of 5 or 0.05. The supernatant was harvested at the indicated times and viral titers were determined by TCID50 assay. Results are means from triplicate samples. Error bars represented standard deviations. ns = not significant. (C) WT and IL16 KO mice were intranasally infected with 5×10 4 PFU of MHV68. Lungs of infected mice were collected at day 4 and 7 post-infection. Virus titers were determined by TCID50 assay. Data represented one of two independent experiments with 5 or 7 mice per group. ns = not significant. Each symbol represented an individual mouse. The horizon line indicated geometric mean titer. (D) Vector or IL16-expressing plasmids with Flag tag were transfected into BHK21 cells for 24 hr, followed by MHV68 infection at an MOI of 5 or 0.05. The infected cells were harvested at the indicated time points and immunoblot analyses were performed with specific antibodies as indicated.–and + represents the cells transfected with vector and IL16-expressing plasmids with Flag tag, respectively. (E) Supernatant was harvested at the indicated times and viral titers were determined by TCID50 assay. Results were means from triplicate samples. Error bars represented standard deviations. ns = not significant.

Article Snippet: Mouse IL16 Antibody (842373) was from R&D Systems.

Techniques: Infection, Western Blot, Control, TCID50 Assay, Virus, Plasmid Preparation, Expressing, FLAG-tag, Transfection

Journal: PLoS Pathogens

Article Title: Interleukin 16 contributes to gammaherpesvirus pathogenesis by inhibiting viral reactivation

doi: 10.1371/journal.ppat.1008701

Figure Lengend Snippet: (A) WT and three IL16 KO single clones were stimulated with (+) or without (-) anti-mouse Ig(G+M) (5 μg/mL) for 48 hr, respectively. Immunoblot analyses were performed with the indicated antibodies. GAPDH was used as a loading control. (B) WT and IL16 KO cells (clone E6) were stimulated with (+) or without (-) anti-mouse Ig(G+M) (5 μg/mL) for 48 hr. Immunoblot analyses were performed with the indicated antibodies. GAPDH was used as a loading control. MHV68 viral genome was determined by qPCR with the primers specific to the MHV68 ORF50 coding region. The relative copy of the MHV68 viral genome was normalized to GAPDH in each sample. (C) The mRNA expression of MHV68 viral gene ORF73, ORF50, ORF59, and ORF25 was determined by qRT-PCR. The relative RNA amount was normalized to GAPDH in each sample. Histograms represented the mean of three independent biological replicates ±SD, p value was determined by two-tailed unpaired t-test, p ≤ 0.05 represents significance.

Article Snippet: Mouse IL16 Antibody (842373) was from R&D Systems.

Techniques: Clone Assay, Western Blot, Control, Expressing, Quantitative RT-PCR, Two Tailed Test

Journal: PLoS Pathogens

Article Title: Interleukin 16 contributes to gammaherpesvirus pathogenesis by inhibiting viral reactivation

doi: 10.1371/journal.ppat.1008701

Figure Lengend Snippet: (A) The diagram showed the potential cleavage sites of IL16. (B) 293T cells were transfected with IL16 or mutants with Flag tag. At 48 hr post-transfection, the supernatant was collected and subjected to IL16 ELISA assay; whole-cell lysates (WCL) and supernatant (SUP) were prepared and subjected to immunoblot analyses with the indicated antibodies. (C) IL16 KO SL-1 cells were transfected with vector (Vec), IL16-, or IL16(D516A)-expressing plasmid with Flag tag, followed by anti-mouse Ig(G+M) treatment for 48 hr. The whole-cell lysates were prepared and subjected to immunoblot analyses with the indicated antibodies.

Article Snippet: Mouse IL16 Antibody (842373) was from R&D Systems.

Techniques: Transfection, FLAG-tag, Enzyme-linked Immunosorbent Assay, Western Blot, Plasmid Preparation, Expressing

Journal: PLoS Pathogens

Article Title: Interleukin 16 contributes to gammaherpesvirus pathogenesis by inhibiting viral reactivation

doi: 10.1371/journal.ppat.1008701

Figure Lengend Snippet: WT and IL16 KO mice were inoculated intranasally with 5×10 4 PFU of MHV68-H2bYFP. Mice inoculated with 5×10 4 PFU of WT MHV68 were used as a control to gate YFP+ cells. Splenocytes were isolated at day 16 and day 18 post-infection. (A) Representative flow plots showing the identification of MHV68-infected YFP+ cells. (B) Frequency of YFP+ cells at day 16 post-infection. Results were compiled from two independent experiments with 8–9 mice per group. Each symbol represented an individual mouse, and the horizon lines represented the mean frequency of infected cells. ns = not significant. (C) Frequency of splenocytes capable of reactivating virus by ex-vivo assay at day 16 post-infection. Serial dilutions of splenocytes were plated on MEFs and the presence of reactivating virus was determined by the presence of cytopathic effect (CPE). Representative results were from two independent experiments with 8–9 mice per group. (D) Frequency of YFP+ cells at day 18 post-infection. Results were compiled from two independent experiments with 10–12 mice per group. Each symbol represented an individual mouse, and the horizon lines represented the mean frequency of infected cells. ns = not significant. (E) Frequency of splenocytes capable of reactivating virus by ex-vivo assay at day 18 post-infection. Data were generated from two independent experiments, 5 to 6 mice per experiment per group.

Article Snippet: Mouse IL16 Antibody (842373) was from R&D Systems.

Techniques: Control, Isolation, Infection, Virus, Ex Vivo, Generated

Journal: PLoS Pathogens

Article Title: Interleukin 16 contributes to gammaherpesvirus pathogenesis by inhibiting viral reactivation

doi: 10.1371/journal.ppat.1008701

Figure Lengend Snippet: WT and IL16 KO mice were intranasally inoculated with 5×10 4 PFU of MHV68-H2bYFP and splenocytes were harvested at day 16 post-infection. (A) Representative flow plots showing the identification of MHV68-infected YFP+ cells. (B) Representative flow plots of YFP+ germinal center B cells (CD19 + CD95 + GL-7 + YFP + ). (C) Quantitation of the percentage of YFP+ germinal center B cells. Each symbol represented an individual mouse, and the horizon lines represented the mean frequency. (D) Representative flow plots of YFP+ plasma cells (CD3 - YFP + B220 low CD138 + ). (E) Quantitation of the percentage of YFP+ plasma cells. Each symbol represented an individual mouse, and the horizon lines represented the mean frequency. (F) Quantitation of the percentage of total germinal center B cells (CD19 + CD95 + GL-7 + ). (G) Quantitation of the percentage of total plasma cells (CD3 - B220 low CD138 + ). ns = not significant. p value was determined by two-tailed unpaired t-test, p ≤ 0.05 represents significance.

Article Snippet: Mouse IL16 Antibody (842373) was from R&D Systems.

Techniques: Infection, Quantitation Assay, Clinical Proteomics, Two Tailed Test

Journal: PLoS Pathogens

Article Title: Interleukin 16 contributes to gammaherpesvirus pathogenesis by inhibiting viral reactivation

doi: 10.1371/journal.ppat.1008701

Figure Lengend Snippet: WT and IL16 KO mice were inoculated intranasally with 5×10 4 PFU of MHV68-H2bYFP. Splenocytes were isolated at day 16 post-infection and subjected to flow cytometry analyses. (A) Representative flow plots of CD4+ and CD8+ T cells from infected WT and IL16 KO mice. (B) Quantitation of the percentage of CD4+ and CD8+ T cells. Each symbol represented an individual mouse, and the horizon lines represented the mean frequency. (C) Representative flow plots of IFN-γ+CD4+ T cells from infected WT and IL16 KO mice. (D) Quantitation of the percentage of IFN-γ+CD4+ and CD44+CD4+ T cells. Each symbol represented an individual mouse, and the horizon lines represented the mean frequency. (E) Quantitation of the percentage of IL4+CD4+ and IL2+CD4+ T cells. Each symbol represented an individual mouse, and the horizon lines represented the mean frequency. (F) Representative flow plots of IFN-γ+ CD8+ T cells from infected WT and IL16 KO mice. (G) Quantitation of the percentage of IFN-γ+CD8+ and TNF-α+CD8+ T cells. Each symbol represented an individual mouse, and the horizon lines represented the mean frequency. ns = not significant. p value was determined by two-tailed unpaired t-test, p ≤ 0.05 represents significance.

Article Snippet: Mouse IL16 Antibody (842373) was from R&D Systems.

Techniques: Isolation, Infection, Flow Cytometry, Quantitation Assay, Two Tailed Test

Journal: PLoS Pathogens

Article Title: Interleukin 16 contributes to gammaherpesvirus pathogenesis by inhibiting viral reactivation

doi: 10.1371/journal.ppat.1008701

Figure Lengend Snippet: (A) The murine M12 B lymphoma cells were transfected with renilla reporter, a luciferase reporter (pGL2) driven by RTA proximal promoter (RTAp), together with IL16-expressing plasmid with Flag tag or vector alone (Vec). Luciferase activity was normalized to renilla activity and Luciferase value was reported as fold increase in luciferase activity over basal promoter activity. Each sample was done in triplicate (two independent experiments). IL16 expression was detected by immunoblot with a Flag antibody. (B) M12 cells were transfected with RTAp together with vector (Vec), IL16-, IL16(D506A)- or IL16(D516A)-expressing plasmid with Flag tag. At 24 hr post-transfection, transfected cells were treated with (+) or without (-) anti-mouse Ig(G+M) (5 μg/mL) for 12 hr. IL16, IL16 (D506A), and IL16 (D516A) expression was detected by immunoblot with Flag antibody. Luciferase value was reported as fold increase in luciferase activity over basal promoter activity. Each sample was done in triplicate (two independent experiments).

Article Snippet: Mouse IL16 Antibody (842373) was from R&D Systems.

Techniques: Transfection, Luciferase, Expressing, Plasmid Preparation, FLAG-tag, Activity Assay, Western Blot

Journal: PLoS Pathogens

Article Title: Interleukin 16 contributes to gammaherpesvirus pathogenesis by inhibiting viral reactivation

doi: 10.1371/journal.ppat.1008701

Figure Lengend Snippet: (A) WT and IL16 KO SL-1 cells were treated with (+) or without (-) 20 μM JAKs inhibitor AG490 for 1 hr, followed by stimulation with (+) or without (-) anti-mouse Ig(G+M) (5 μg/mL) for 48 hr. Immunoblot analyses were performed with specific antibodies as indicated. GAPDH was used as a loading control. (B) WT and IL16 KO SL-1 cells were treated with (+) or without (-) anti-mouse Ig(G+M) (5 μg/mL) for 48 hr. Immunoblot analyses were performed with specific antibodies as indicated. (C) Quantitation of phosphorylated STAT relative to total STAT based on immunoblot detection in C using the ImageJ image analysis software.

Article Snippet: Mouse IL16 Antibody (842373) was from R&D Systems.

Techniques: Western Blot, Control, Quantitation Assay, Software

Journal: PLoS Pathogens

Article Title: Interleukin 16 contributes to gammaherpesvirus pathogenesis by inhibiting viral reactivation

doi: 10.1371/journal.ppat.1008701

Figure Lengend Snippet: (A) WT and IL16 KO SL-1 cells were pretreated with DMSO, 40 μM, or 80 μM STAT5 inhibitor (STAT5-I) for 1 hr, followed by stimulation with anti-mouse Ig(G+M) (5 μg/mL) for 48 hr. Immunoblot analyses were performed with specific antibodies as indicated. (B) SL-1 cells were transfected with vector, STAT3-expressing plasmid, or STAT3C-expressing plasmid with Flag tag, followed by anti-mouse Ig(G+M) (5 μg/mL) treatment for 48 hr. Immunoblot analyses were performed with the indicated antibodies. (C) WT and IL16 KO SL-1 cells were treated with (+) or without (-) anti-mouse Ig(G+M) (5 μg/mL) for 48 hr. Immunoblot analyses were performed with the specific antibodies as indicated. (D) Quantitation of phosphorylated STAT3(Y705) relative to total STAT3 and p21 relative to GAPDH based on immunoblot detection in C using the ImageJ image analysis software. (E) WT and IL16 KO SL-1 cells were treated with DMSO or Orthovanadate (50 μM) in the presence or absence of anti-mouse Ig(G+M) (5 μg/mL) for 48 hr. Immunoblot analyses were performed with the specific antibodies as indicated.

Article Snippet: Mouse IL16 Antibody (842373) was from R&D Systems.

Techniques: Western Blot, Transfection, Plasmid Preparation, Expressing, FLAG-tag, Quantitation Assay, Software

Journal: PLoS Pathogens

Article Title: Interleukin 16 contributes to gammaherpesvirus pathogenesis by inhibiting viral reactivation

doi: 10.1371/journal.ppat.1008701

Figure Lengend Snippet: MHV68 infection induces IL16 production, which, in turn, increases STAT3(Y705) phosphorylation, subsequently reduces p21 expression, and inhibits MHV68 reactivation. Meanwhile , IL16 partially inhibits RTA promoter activity and STAT3(S727) phosphorylation, contributing to the inhibition of MHV68 reactivation. Ultimately, MHV68-induced IL16 helps to maintain MHV68 latency.

Article Snippet: Mouse IL16 Antibody (842373) was from R&D Systems.

Techniques: Infection, Phospho-proteomics, Expressing, Activity Assay, Inhibition