Journal: Oncogene

Article Title: F box protein FBXL2 exerts human lung tumor suppressor-like activity by ubiquitin-mediated degradation of cyclin D3 resulting in cell cycle arrest

doi: 10.1038/onc.2011.432

Figure Lengend Snippet: A. MLE cells were transfected with either FBXL2 or mutant FBXL2 lacking the NH 2 -terminal 100 residues. Cells were then observed under white field microscopy or assayed for ATP production. B. FACS analysis in MLE cells transfected with either an empty vector or FBXL2 plasmid (left panels, and upper right panel, n=3 ). DNA replication assay, monitored by BrdU incorporation (right lower panel, error bars indicate S.D.; n =3, *P<0.01). C. Immunoblotting showing levels of cyclins, CDKs, and negative control proteins, actin, Lpcat, and Erk, and a positive control protein (cytidylyltransferase, [CCT]), after control (CON) plasmid or ectopic FBXL2 expression. D. FACS analysis in cells transfected with scrambled RNA or siRNA to cyclin D2, cyclin D3, or both targets (lower panel). Immunoblotting of cells for cyclins and β-actin from lysates (upper panel). E. Cyclin D3 protein half-life determination after FBXL2 overexpression ( E ), or FBXL2 knockdown using siRNA ( n =3 experiments). Below each panel levels of each protein on immunoblots were quantified densitometrically and shown graphically.

Article Snippet: Mouse monoclonal cyclin D2 and cyclin D3 antibodies were from Abcam (Cambridge, MA).

Techniques: Transfection, Mutagenesis, Microscopy, Plasmid Preparation, BrdU Incorporation Assay, Western Blot, Negative Control, Positive Control, Control, Expressing, Over Expression, Knockdown

Journal: Oncogene

Article Title: F box protein FBXL2 exerts human lung tumor suppressor-like activity by ubiquitin-mediated degradation of cyclin D3 resulting in cell cycle arrest

doi: 10.1038/onc.2011.432

Figure Lengend Snippet: A. MLE cells were synchronized to each cell phase followed by co-immunoprecipitation of endogenous FBXL2 and then cyclin D3 immunoblotting. Shown on left are steady-state levels of cyclin D3, FBXL2, and actin in cell lysates. B. Cells were also immunostained for cyclin D3 and counterstained with DAPI to visualize nucleus. White arrows indicate at mitotic cells. Fluorescent intensity of endogenous cyclin D3 levels within cells at interphase versus cells undergoing mitosis was quantified using imageJ software and graphed on the right panel. C . In vivo ubiquitination assays. Polyubiquitinated cyclin D3 was detected by immunoprecipitation of endogenous cyclins followed by immunoblotting for ubiquitin. The arrows show polyubiquitinated cyclin D3. D. Cyclin D3 levels in cells treated with leupeptin or MG132. E. In vitro ubiquitination assays. Purified SCF complex components were incubated with V5-cyclin D3 and the full complement of ubiquitination reaction components (second lane from left) showing polyubiquitinated cyclin D3.

Article Snippet: Mouse monoclonal cyclin D2 and cyclin D3 antibodies were from Abcam (Cambridge, MA).

Techniques: Immunoprecipitation, Western Blot, Software, In Vivo, Ubiquitin Proteomics, In Vitro, Purification, Incubation

Journal: Oncogene

Article Title: F box protein FBXL2 exerts human lung tumor suppressor-like activity by ubiquitin-mediated degradation of cyclin D3 resulting in cell cycle arrest

doi: 10.1038/onc.2011.432

Figure Lengend Snippet: A. Primary sequence of cyclin D3. Red rectangle represents a potential IQ motif within cyclin D3. Red arrow indicates a potential ubiquitination site within cyclin D3. Blue arrow indicates a potential phosphorylation site within cyclin D3. B. Immunoblotting for accumulation of cyclin D3 wild type (WT) or point mutants in cells in the absence (−) or presence (+) of MG132 treatment. The arrows indicate lack of polyubiquitinated signals after expression of a Lys 268R cyclin D3 mutant. C. Cyclin D3 protein half-life determination after expression of WT V5-cyclin D3, or Lys 268R (V5-cyclin D3) mutant (data are from n =2 experiments). Below each panel levels of each protein on immunoblots were quantified densitometrically and shown graphically. D. Cyclin D3 protein levels in cells after co-transfection with either WT cyclin D3, or Lys 268R cyclin D3 with or without ectopic FBXL2 expression. E. In vitro ubiquitination assays. Purified SCF complex were incubated with WT V5-cyclin D3, or Lys 268R V5-cyclin D3 mutant and the full complement of ubiquitination reaction components. F. FACS analysis in cells prepared in D ( n =3 experiments).

Article Snippet: Mouse monoclonal cyclin D2 and cyclin D3 antibodies were from Abcam (Cambridge, MA).

Techniques: Sequencing, Ubiquitin Proteomics, Phospho-proteomics, Western Blot, Expressing, Mutagenesis, Cotransfection, In Vitro, Purification, Incubation

Journal: Oncogene

Article Title: F box protein FBXL2 exerts human lung tumor suppressor-like activity by ubiquitin-mediated degradation of cyclin D3 resulting in cell cycle arrest

doi: 10.1038/onc.2011.432

Figure Lengend Snippet: A. CaM-sepharose pull-down (PD) assays showing effects of exogenous calcium on binding between CaM and either V5-full-length (FL) cyclins or V5-NH 2 -terminal truncated (N100) mutants lacking the IQ motif within cyclin D3 (upper panel). CaM-sepharose pull-down assays showing levels of binding between CaM and WT cyclin D3 or D3 variants harboring point mutations within IQ motifs (lower panel). B. Coimmunoprecipitation of endogenous FBXL2 and V5-immunoblotting for FL or NH 2 -terminal truncated (N100) mutant cyclin D3 (upper panel). Cells were transfected with V5-cyclin D3 variants harboring point mutations within IQ motifs followed by co-immunoprecipitation of endogenous FBXL2 and V5-immunoblotting (lower panel). C−D. Cells were co-transfected with WT cyclin D3 or a variant harboring a point mutation within the IQ motif with or without FBXL2 plasmid followed by immunoblotting for cyclins (left blot). D . Right: Cells were also analyzed by FACS, ( n =3 experiments). E. In vitro ubiquitination assays. Purified SCF complex were incubated with WT V5-cyclin D3, or an IQ motif point mutant, and the full complement of ubiquitination reaction components. F. Cyclin D3 protein half-life determination after expression of WT V5-cyclin D3, or an IQ motif point mutant ( n =2 experiments). Below the panels levels of each protein on immunoblots were quantified densitometrically and shown graphically. G. Cell lysates expressing his-V5-FBXL2 truncation mutants (top, map) co-purified with GST-cyclin D3 using his-PD. After washing, proteins were eluted and processed for V5 or cyclin D3 immunoblotting.

Article Snippet: Mouse monoclonal cyclin D2 and cyclin D3 antibodies were from Abcam (Cambridge, MA).

Techniques: Binding Assay, Western Blot, Mutagenesis, Transfection, Immunoprecipitation, Variant Assay, Plasmid Preparation, In Vitro, Ubiquitin Proteomics, Purification, Incubation, Expressing

Journal: Oncogene

Article Title: F box protein FBXL2 exerts human lung tumor suppressor-like activity by ubiquitin-mediated degradation of cyclin D3 resulting in cell cycle arrest

doi: 10.1038/onc.2011.432

Figure Lengend Snippet: A. Cyclin D3 protein half-life determination after adenoviral (Adv) CaM overexpression, or CaM knockdown using siRNA ( n =2 experiments). Below each panel levels of each protein on immunoblots were quantified densitometrically and shown graphically. B. FACS analysis of cells after CaM overexpression or knockdown, ( n =2 experiments). C. Levels of endogenous cyclin D3 protein in cells after co-expression of either Adv-empty, Adv-CaM, or WT FBXL2 or a FBXL2 mutant (F79A) that lacks ability to bind CaM. D. FACS analysis of cells prepared in C , ( n =3 experiments). E. MLE cells were synchronized to each cell phase, followed by co-immunoprecipitation of endogenous FBXL2 or cyclin D3, and immunoblotting for CaM. Top immunoblot: input of CaM in total cell lysates prior to i.p. F. V5-FBXL2-agarose beads were generated and used as bait and incubated with combinations of purified GST-cyclin D3, or CaM with or without exogenous calcium. After washing of beads (150mM NaCl, 0.1% Triton X-100), proteins were eluted and resolved by SDS-PAGE followed by cyclin D3, CaM, and V5 immunoblotting. G. ITC binding analysis of CaM and peptide (LQLLGTVCLL) encoding a CaM-binding motif within cylicn D3 in vitro .

Article Snippet: Mouse monoclonal cyclin D2 and cyclin D3 antibodies were from Abcam (Cambridge, MA).

Techniques: Over Expression, Knockdown, Western Blot, Expressing, Mutagenesis, Immunoprecipitation, Generated, Incubation, Purification, SDS Page, Binding Assay, In Vitro

Journal: Oncogene

Article Title: F box protein FBXL2 exerts human lung tumor suppressor-like activity by ubiquitin-mediated degradation of cyclin D3 resulting in cell cycle arrest

doi: 10.1038/onc.2011.432

Figure Lengend Snippet: A–B. FACS analysis showing levels of apoptotic human adenocarcinoma (A549) cells after FBXL2 overexpression, ( n=3 experiments, * P <0.01 versus empty). C. Proliferation studies of A549 cells after FBXL2 overexpression, ( n=3 experiments, * P <0.01 versus FBXL2). D. Proliferation studies of A549 cells after FBXL2 or CaM knockdown ( n=3 experiments, * P <0.05 versus control [CON]). E. A549 cells were treated with four chemotherapeutic agents using low (L) or high (H) concentrations. 18 h later, cells were collected, lysed, and immunoblotted with antibodies to the indicated proteins. F. FBXL2 and cyclin D3 immunoreactive levels after treatment with the chemotherapeutic agent vinorelbine. G. FACS analysis showing levels of apoptotic human adenocarcinoma (A549) cells after FBXL2 or CaM knockdown with vinorelbine treatment ( n=3 experiments, * P <0.01 versus Con RNA, # P <0.01 versus 0 h). H. Data is quantated from FACS above.

Article Snippet: Mouse monoclonal cyclin D2 and cyclin D3 antibodies were from Abcam (Cambridge, MA).

Techniques: Over Expression, Knockdown, Control

Journal: Oncogene

Article Title: F box protein FBXL2 exerts human lung tumor suppressor-like activity by ubiquitin-mediated degradation of cyclin D3 resulting in cell cycle arrest

doi: 10.1038/onc.2011.432

Figure Lengend Snippet: A. Wound-healing assay. Confluent monolayers of A549 cells were injured and cellular migration into the wound was determined under control (CON) conditions, after FBXL2 overexpression (left panels), or after ectopic expression of cyclin D3 variants that harbor mutations within the ubiquitin acceptor site (K mutant, top right panel) or within the IQ motif (bottom right panel). Recovery of cells to wound heal was quatitated and graphed (right) * P <0.01 versus other groups. B. FACS analysis showing levels of apoptotic A549 cells after doxycycline (Dox) inducible FBXL2 overexpression, ( n=3 experiments, * P <0.05 versus 0 hr). The immunoblot on right shows levels of indicated proteins in cells. C–D. Effect of stable expression of FBXL2 or a control vector (Empty) on growth of A549 tumor implants in nude mice, n=8 mice/group. The upper left panel shows tumor volume over time and shown at right are representative images of variable sizes of xenografts in two nude mice (arrows) after expression of an empty vector or FBXL2. For C, n=8 mice/group * P <0.05 versus FBXL2. In (D), tumors from four control and four FBXL2 treated A549 implants in mice were collected at the end-point, and assayed for cyclin D3 and FBXL2 proteins by immunoblotting. The right bar graph shows the denistometric data from immunoblots, * P <0.05 versus empty.

Article Snippet: Mouse monoclonal cyclin D2 and cyclin D3 antibodies were from Abcam (Cambridge, MA).

Techniques: Wound Healing Assay, Migration, Control, Over Expression, Expressing, Ubiquitin Proteomics, Mutagenesis, Western Blot, Plasmid Preparation

Journal: Oncogene

Article Title: F box protein FBXL2 exerts human lung tumor suppressor-like activity by ubiquitin-mediated degradation of cyclin D3 resulting in cell cycle arrest

doi: 10.1038/onc.2011.432

Figure Lengend Snippet: A. Calmodulin (CaM) binds within an IQ signature to protect cyclin D3 from SCF FBXL2 . This interaction is calcium independent and preserves cyclin D3 interaction with Cdk11p58 that promotes mitosis by recruitment of PLK4 and Aurora A to the centrosomes (bottom, left). B . Increases in calcium promotes SCF FBXL2 competing with CaM for IQ motif binding within cyclin D3 to mediate cyclin D3 polyubiquitination and subsequent degradation compromising its association with and activity of CDk11p58, essential for cell cycle progression. This process dislocates PLK4 and Aurora A from the centrosome, resulting in cell cycle arrest. C . In addition to competition for cyclin D IQ motif binding, CaM directly binds and antagonizes SCF FBXL2 .

Article Snippet: Mouse monoclonal cyclin D2 and cyclin D3 antibodies were from Abcam (Cambridge, MA).

Techniques: Binding Assay, Activity Assay

Journal: Oncotarget

Article Title: Allergen specific immunotherapy enhanced defense against bacteria via TGF-β1-induced CYP27B1 in asthma

doi: 10.18632/oncotarget.19826

Figure Lengend Snippet: (A) Frozen lung sections were stained with polyclonal antibodies against CRAMP followed by incubation with FITC-conjugated secondary antibody (green) or PI (red). Sections were observed using confocal microscopy (original magnification×400). Results are representative of three experiments. (B) The mRNA expression of CRAMP was determined in the lungs using Real-time-PCR. (C) Standard ELISA was performed to determine the levels of CRAMP levels in the BAL fluid. (D) Lung sections were stained with polyclonal antibodies against CYP27B1. Sections were observed using microscopy (original magnification×200). (E) The mRNA expression of CYP27B1 was determined in the lungs using Real-time-PCR. Data were shown as means±SDEV (n=8). One-way ANOVA for group comparisons. Significant differences in the mean values were defined as p < 0.05.

Article Snippet: The membranes was incubated overnight at 4°C with rabbit polyclonal antibody against CRAMP (1:1000 diluted with 1% skim milk, PAB13021, Abnova, Taipei), or rabbit polyclonal antibody against CYP27b1 (1:200 diluted with 1% skim milk, sc-67261, Santa Cruz Biotechnology, USA), or rabbit monoclonal antibody against GAPDH (1:1000 diluted with 1% skim milk, Cat. No. 2118, Cell Signaling, USA).

Techniques: Staining, Incubation, Confocal Microscopy, Expressing, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Microscopy

Journal: Oncotarget

Article Title: Allergen specific immunotherapy enhanced defense against bacteria via TGF-β1-induced CYP27B1 in asthma

doi: 10.18632/oncotarget.19826

Figure Lengend Snippet: (A) Western blot analysis of CRAMP with 16HBE cells exposed to TGF-β1, 25VD 3 , TGF-β1/25VD 3 and ITRA/TGF-β1/25VD 3. Relative density (change) of CRAMP in cells. β-actin was used as loading control. (B) Western blot analysis of CYP27B1 in 16HBE cells exposed to TGF-β1. Relative density (change) of CRAMP in cells. β-actin was used as loading control. The mRNA expression of CRAMP (C) and CYP27B1 (D) was determined in cells using Real-time-PCR. Results are representative of three experiments and data are shown as means± SDEV. One-way ANOVA for group comparisons. Significant differences in the mean values were defined as p < 0.05.

Article Snippet: The membranes was incubated overnight at 4°C with rabbit polyclonal antibody against CRAMP (1:1000 diluted with 1% skim milk, PAB13021, Abnova, Taipei), or rabbit polyclonal antibody against CYP27b1 (1:200 diluted with 1% skim milk, sc-67261, Santa Cruz Biotechnology, USA), or rabbit monoclonal antibody against GAPDH (1:1000 diluted with 1% skim milk, Cat. No. 2118, Cell Signaling, USA).

Techniques: Western Blot, Control, Expressing, Real-time Polymerase Chain Reaction

Journal: Oncotarget

Article Title: Allergen specific immunotherapy enhanced defense against bacteria via TGF-β1-induced CYP27B1 in asthma

doi: 10.18632/oncotarget.19826

Figure Lengend Snippet: (A) 16HBE cells were pretreated with TGF-β1 and TGF-β1/25VD 3 . (B) 16HBE cells were pretreated with ITRA. The cells infected with P. aeruginosa cells were lysed and plated to confirm the presence of intracellular bacteria. Results are representative of three experiments and data are shown as means±SDEV. One-way ANOVA for group comparisons. Significant differences in the mean values were defined as p < 0.05. (C) Schematic representation of TGF-β1 induced CYP27B1 role in HDM immunotherapy increasing defense against P. aeruginosa during allergic airway inflammation.

Article Snippet: The membranes was incubated overnight at 4°C with rabbit polyclonal antibody against CRAMP (1:1000 diluted with 1% skim milk, PAB13021, Abnova, Taipei), or rabbit polyclonal antibody against CYP27b1 (1:200 diluted with 1% skim milk, sc-67261, Santa Cruz Biotechnology, USA), or rabbit monoclonal antibody against GAPDH (1:1000 diluted with 1% skim milk, Cat. No. 2118, Cell Signaling, USA).

Techniques: Infection, Bacteria

Journal: Nucleic Acids Research

Article Title: Condensin I and condensin II proteins form a LINE-1 dependent super condensin complex and cooperate to repress LINE-1

doi: 10.1093/nar/gkac802

Figure Lengend Snippet: Condensin I represses L1 retrotransposition and expression. ( A ) Schematic of the condensin I complex; the names of condensin I subunits are indicated by the different colored shapes. ( B–D ) HT-29 cells were induced to express either Non-target, control shRNA or NCAPD2 shRNA. (B) Cellular RNAs were analyzed by qRT-PCR to detect NCAPD2 transcript levels. Transcripts were normalized to actin transcript levels; the averages of three independent experiments are shown. Whole cell lysates were subjected to immunoblot analysis to detect (C) NCAPD2 protein or (D) endogenous ORF1p. Quantification of three independent experiments are shown to the right of each blot in panels C and D. Band intensities were quantified from film and normalized to actin. ( E ) HT-29 cells were induced to express either Non-Target shRNA or NCAPD2 shRNA and then were transfected with equal amounts of a pJM101/L1.3 expression vector to assess retrotransposition efficiency. Quantification of three independent experiments is shown. ( F–H ) HT-29 cells were induced to express either Non-target, control shRNA or NCAPG shRNA. (F) Cellular RNAs were analyzed by qRT-PCR to detect NCAPG transcript levels. Transcripts were normalized to actin transcript levels, and the averages of three independent experiments are shown. Whole cell lysates were subjected to immunoblot analysis to detect (G) NCAPG protein or (H) endogenous ORF1p. Quantification of three independent experiments is shown to the right of each blot in panels G and H. Band intensities were quantified from film and normalized to actin. ( I ) HT-29 cells were induced to express either Non-Target shRNA or NCAPG shRNA and then were transfected with equal amounts of pJM101/L1.3 to assess retrotransposition efficiency. Quantification of three independent experiments is shown. P values were determined by performing unpaired t -tests. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001. Error bars indicate standard deviations from the mean.

Article Snippet: The following primary antibodies were utilized throughout this study for immunoblot analyses, immunoprecipitation experiments, or Proximity Ligation Assays: NCAPD3 (Bethyl Laboratories, catalog #A300-604A), NCAPD2 (Bethyl Laboratories, catalog #A300-601A and Santa Cruz Biotechnology, catalog #sc-166878), NCAPG2 (Abcam, catalog #ab70350), NCAPG (Bethyl Laboratories, catalog #A300-602A), EPRS (Bethyl Laboratories, catalog #A303-957A), eIF4G (Santa Cruz Biotechnology, catalog #sc-11373), Actin (Millipore, catalog #MAB1501), Normal Rabbit IgG (Millipore, catalog #12–370), SMC4 (Bethyl Laboratories, catalog #A300-063A), SMC2 (Bethyl Laboratories, catalog #A300-058A), NCAPH2 (Bethyl Laboratories, catalog #A302-276A), β-tubulin (Cell Signaling, catalog #2146).

Techniques: Expressing, Control, shRNA, Quantitative RT-PCR, Western Blot, Transfection, Plasmid Preparation

Journal: Nucleic Acids Research

Article Title: Condensin I and condensin II proteins form a LINE-1 dependent super condensin complex and cooperate to repress LINE-1

doi: 10.1093/nar/gkac802

Figure Lengend Snippet: NCAPD2/ condensin I is required for EPRS and NCAPD3 association with L1 RNA. ( A ) HT-29 cells were transfected with either pJM101/L1.3 expression vector or a L1 construct harboring a deletion within its 3′UTR (pJM101/L1.3 3′UTRΔ) and RIP/qRT-PCR assays were conducted using a NCAPD2 antibody to immunoprecipitate L1 RNA. Reactions lacking an antibody (No Antibody IP) and reactions involving a non-specific, IgG antibody served as negative controls. Sequences within the neomycin resistance cassette present in pJM101/L1.3 and pJM101/L1.3 3′UTRΔ were used to design qRT-PCR primers to detect L1 RNA. The average levels of RNA-bound protein were calculated as percentages of the input used for each immunoprecipitation; averages from three independent experiments are shown. ( B ) EPRS co-IP/immunoblot experiments were conducted in HT-29 cells to detect association with NCAPD2. IPs using antibody only (ie. no lysate) and IPs using an IgG antibody served as negative controls. A representative blot of three independent experiments is shown. ( C–E ) HT-29 cells were induced to express either NT shRNA or NCAPD2 shRNA and then were transfected with pJM101/L1.3. RIP/qRT-PCR assays were conducted using either (C) NCAPD3 antibody, (D) EPRS antibody, or (E) eIF4G antibody to immunoprecipitate L1 RNA, as described for (A). P values were determined by performing unpaired t -tests. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001. Error bars indicate standard deviations from the mean.

Article Snippet: The following primary antibodies were utilized throughout this study for immunoblot analyses, immunoprecipitation experiments, or Proximity Ligation Assays: NCAPD3 (Bethyl Laboratories, catalog #A300-604A), NCAPD2 (Bethyl Laboratories, catalog #A300-601A and Santa Cruz Biotechnology, catalog #sc-166878), NCAPG2 (Abcam, catalog #ab70350), NCAPG (Bethyl Laboratories, catalog #A300-602A), EPRS (Bethyl Laboratories, catalog #A303-957A), eIF4G (Santa Cruz Biotechnology, catalog #sc-11373), Actin (Millipore, catalog #MAB1501), Normal Rabbit IgG (Millipore, catalog #12–370), SMC4 (Bethyl Laboratories, catalog #A300-063A), SMC2 (Bethyl Laboratories, catalog #A300-058A), NCAPH2 (Bethyl Laboratories, catalog #A302-276A), β-tubulin (Cell Signaling, catalog #2146).

Techniques: Transfection, Expressing, Plasmid Preparation, Construct, Quantitative RT-PCR, Immunoprecipitation, Co-Immunoprecipitation Assay, Western Blot, shRNA

Journal: Nucleic Acids Research

Article Title: Condensin I and condensin II proteins form a LINE-1 dependent super condensin complex and cooperate to repress LINE-1

doi: 10.1093/nar/gkac802

Figure Lengend Snippet: NCAPD2 and NCAPD3 associate to form SCCs in a L1 RNA dependent manner. ( A ) Reciprocal NCAPD3 and NCAPD2 co-IP/immunoblots were conducted in HT-29 cells to detect an association between NCAPD3 and NCAPD2. Antibody only (i.e. no lysate) and IgG antibody IPs served as negative controls. Each experiment was performed three times and representative blots are shown. ( B ) NCAPG2 co-IP/immunoblot experiments were conducted in HT-29 cells to detect an association with NCAPD2 (middle panel) and with NCAPG (bottom panel). Antibody only (i.e. no lysate) and IgG antibody IPs served as negative controls. Each experiment was performed three times and representative blots are shown. ( C ) Proximity Ligation Assays (PLAs) were performed to detect an association between NCAPD2 and NCAPD3 in HT-29 cells transfected with control (siCTRL) or L1 siRNA (siL1). Single antibody controls were performed in parallel for each experiment. Images were taken using a confocal microscope with a 63x objective; maximum projections are shown. ( D ) Volocity imaging software was used to analyze confocal images and quantify the average number of nuclear and cytoplasmic PLA foci per cell. Each dot represents the average of 50–150 nuclei evaluated from a single image. Images were taken from three independent experiments. ( E ) PLAs were performed to detect associations between NCAPD3 and NCAPG2 in HT-29 cells transfected with siCTRL or siL1 and results were quantified as described in (D). P values were determined by performing unpaired t -tests. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001, ns = not significant. Error bars indicate standard deviations from the mean.

Article Snippet: The following primary antibodies were utilized throughout this study for immunoblot analyses, immunoprecipitation experiments, or Proximity Ligation Assays: NCAPD3 (Bethyl Laboratories, catalog #A300-604A), NCAPD2 (Bethyl Laboratories, catalog #A300-601A and Santa Cruz Biotechnology, catalog #sc-166878), NCAPG2 (Abcam, catalog #ab70350), NCAPG (Bethyl Laboratories, catalog #A300-602A), EPRS (Bethyl Laboratories, catalog #A303-957A), eIF4G (Santa Cruz Biotechnology, catalog #sc-11373), Actin (Millipore, catalog #MAB1501), Normal Rabbit IgG (Millipore, catalog #12–370), SMC4 (Bethyl Laboratories, catalog #A300-063A), SMC2 (Bethyl Laboratories, catalog #A300-058A), NCAPH2 (Bethyl Laboratories, catalog #A302-276A), β-tubulin (Cell Signaling, catalog #2146).

Techniques: Co-Immunoprecipitation Assay, Western Blot, Ligation, Transfection, Control, Microscopy, Imaging, Software

Journal: Nucleic Acids Research

Article Title: Condensin I and condensin II proteins form a LINE-1 dependent super condensin complex and cooperate to repress LINE-1

doi: 10.1093/nar/gkac802

Figure Lengend Snippet: NRTI Treatment reduces L1 transcripts and L1 retrotransposition events and decreases cytoplasmic SCC formation. ( A ) HT-29 cells were treated with either DMSO or DMSO containing Zidovudine and Didanosine at the indicated drug concentrations for 10 days then fixed and stained with crystal violet (CV) to measure cell toxicity. Shown are averages from three independent experiments. ( B ) Retrotransposition assays were performed using HT-29 cells that were transfected with pJM101/L1.3 and treated with either DMSO or a combination of the nucleoside reverse transcriptase inhibitors (NRTIs) Zidovudine and Didanosine; each drug was at a final concentration of 50 μM in tissue culture media. Quantification of retrotransposition assays from three independent experiments is shown. ( C ) qRT-PCR analysis of endogenous L1 RNA levels (using a 5′UTR primer pair; See and Methods) in HT-29 cells treated with either DMSO or NRTIs. The average relative transcript levels for three independent experiments are shown. ( D ) PLA was performed to detect an association between NCAPD2 and NCAPD3 in HT-29 cells treated with DMSO or NRTIs at a final concentration of 50μM in tissue culture media. Single antibody controls were performed in parallel for each experiment. Images were taken using a confocal microscope with a 63x objective and maximum projections are shown. Volocity imaging software was used to analyze confocal images as noted in Figure to quantify the average number of nuclear (left chart) and cytoplasmic (right chart) PLA foci per cell. P values were determined by performing unpaired t -tests. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, ns = not significant. Error bars indicate standard deviations from the mean.

Article Snippet: The following primary antibodies were utilized throughout this study for immunoblot analyses, immunoprecipitation experiments, or Proximity Ligation Assays: NCAPD3 (Bethyl Laboratories, catalog #A300-604A), NCAPD2 (Bethyl Laboratories, catalog #A300-601A and Santa Cruz Biotechnology, catalog #sc-166878), NCAPG2 (Abcam, catalog #ab70350), NCAPG (Bethyl Laboratories, catalog #A300-602A), EPRS (Bethyl Laboratories, catalog #A303-957A), eIF4G (Santa Cruz Biotechnology, catalog #sc-11373), Actin (Millipore, catalog #MAB1501), Normal Rabbit IgG (Millipore, catalog #12–370), SMC4 (Bethyl Laboratories, catalog #A300-063A), SMC2 (Bethyl Laboratories, catalog #A300-058A), NCAPH2 (Bethyl Laboratories, catalog #A302-276A), β-tubulin (Cell Signaling, catalog #2146).

Techniques: Staining, Transfection, Reverse Transcription, Concentration Assay, Quantitative RT-PCR, Microscopy, Imaging, Software

Journal: Nucleic Acids Research

Article Title: Condensin I and condensin II proteins form a LINE-1 dependent super condensin complex and cooperate to repress LINE-1

doi: 10.1093/nar/gkac802

Figure Lengend Snippet: SCC formation occurs in primary cells expressing L1 transcripts. ( A ) qRT-PCR analysis of L1 transcripts in RPE-1 cells transfected with the pCEP4 empty vector or pJM101/L1.3 expression vector. Sequences within the neomycin resistance cassette present in pJM101/L1.3 were used to design qRT-PCR primers to detect L1 RNA. Transcript levels were normalized to actin and L1 transcript levels in pCEP4 transfected cells were set to 1. Shown is the average of three independent experiments. ( B, C ) IP/immunoblotting experiments for NCAPD2 and NCAPD3 were performed in RPE-1 cells transfected with (B) pCEP4 or (C) pJM101/L1.3. Antibody only (no lysate) and IgG antibody IPs served as controls. Asterisks indicate bands remaining after stripping the membrane that was immunoblotted for NCAPD3. Shown are representative blots of three independent experiments. ( D ) Average amounts of co-precipitated NCAPD2 protein, normalized to the amount of immunoprecipitated NCAPD3 protein, were quantified from the experiments in panels B and C. P values were determined by performing unpaired t -tests. * P ≤ 0.05, ** P ≤ 0.01. Error bars indicate standard deviations from the mean.

Article Snippet: The following primary antibodies were utilized throughout this study for immunoblot analyses, immunoprecipitation experiments, or Proximity Ligation Assays: NCAPD3 (Bethyl Laboratories, catalog #A300-604A), NCAPD2 (Bethyl Laboratories, catalog #A300-601A and Santa Cruz Biotechnology, catalog #sc-166878), NCAPG2 (Abcam, catalog #ab70350), NCAPG (Bethyl Laboratories, catalog #A300-602A), EPRS (Bethyl Laboratories, catalog #A303-957A), eIF4G (Santa Cruz Biotechnology, catalog #sc-11373), Actin (Millipore, catalog #MAB1501), Normal Rabbit IgG (Millipore, catalog #12–370), SMC4 (Bethyl Laboratories, catalog #A300-063A), SMC2 (Bethyl Laboratories, catalog #A300-058A), NCAPH2 (Bethyl Laboratories, catalog #A302-276A), β-tubulin (Cell Signaling, catalog #2146).

Techniques: Expressing, Quantitative RT-PCR, Transfection, Plasmid Preparation, Western Blot, Stripping Membranes, Membrane, Immunoprecipitation

Journal: Nucleic Acids Research

Article Title: Condensin I and condensin II proteins form a LINE-1 dependent super condensin complex and cooperate to repress LINE-1

doi: 10.1093/nar/gkac802

Figure Lengend Snippet: The 3′UTR of L1 RNA antagonizes SCC formation. ( A ) HT-29 cells were transfected with the pCEP4 empty vector, pJM101/L1.3 expression vector, or L1 expression vector harboring a 3′UTR deletion (pJM101/L1.3 3′UTRΔ) and Proximity Ligation Assays were performed to detect association between NCAPD2 and NCAPD3. Single antibody controls were performed in parallel for each experiment. Images shown were taken on a confocal microscope with a 63x objective and maximum projections are shown. ( B , C ) Volocity imaging software was used to analyze confocal images as noted in Figure to quantify the average number of nuclear (B) and cytoplasmic (C) PLA foci per cell. Images shown were taken from two independent experiments. P values were determined by performing unpaired t -tests. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, ns = not significant. Error bars indicate standard deviations from the mean.

Article Snippet: The following primary antibodies were utilized throughout this study for immunoblot analyses, immunoprecipitation experiments, or Proximity Ligation Assays: NCAPD3 (Bethyl Laboratories, catalog #A300-604A), NCAPD2 (Bethyl Laboratories, catalog #A300-601A and Santa Cruz Biotechnology, catalog #sc-166878), NCAPG2 (Abcam, catalog #ab70350), NCAPG (Bethyl Laboratories, catalog #A300-602A), EPRS (Bethyl Laboratories, catalog #A303-957A), eIF4G (Santa Cruz Biotechnology, catalog #sc-11373), Actin (Millipore, catalog #MAB1501), Normal Rabbit IgG (Millipore, catalog #12–370), SMC4 (Bethyl Laboratories, catalog #A300-063A), SMC2 (Bethyl Laboratories, catalog #A300-058A), NCAPH2 (Bethyl Laboratories, catalog #A302-276A), β-tubulin (Cell Signaling, catalog #2146).

Techniques: Transfection, Plasmid Preparation, Expressing, Ligation, Microscopy, Imaging, Software

Journal: Frontiers in Behavioral Neuroscience

Article Title: Increased conditioned place preference for cocaine in high anxiety related behavior (HAB) mice is associated with an increased activation in the accumbens corridor

doi: 10.3389/fnbeh.2014.00441

Figure Lengend Snippet: Colocalization of neuronal markers with EGR1 expression 1 h after cocaine CPP is restricted to D1- and D2-medium spiny neurons . Brains were harvested for double fluorescence immunohistochemistry 1 h after the start of a 15-min cocaine CPP test session. EGR1 immunoreactivity (shown in green) remained restricted to nuclei. All other neuronal markers are shown in red. Colocalization of immunoreactivity is found only in cells positive for the neuronal nuclear protein NeuN (A) In contrast, no colocalization with EGR1 was observed in glial cells. The employed glial markers were glial fibrillary acidic protein for astrocytes (GFAP, B ), myelin basic protein for oligodendrocytes (MBP, C ), and tomato lectin for microglia (TL, D ). Colocalization of immunoreactivity was found only in neurons immunoreactive against an anti-dynorphin antibody (DYN, E ), i.e., most likely dopamine D1 receptor expressing medium spiny neurons (D1-MSNs) or in neurons immunoreactive against an anti-dopamine D2 receptor antibody (DRD2, D2-MSNs, F ). No colocalization with EGR1 was observed in cholinergic interneurons (marker: choline acetyltransferase ChAT, G ) or GABAergic interneurons positive for parvalbumin (PV, H ), neuropeptide Y (NPY, I ) or calretinin (CR, J ) Images were taken with a laser scanning confocal microscope with a magnification of 100× (bar size, 10 μm).

Article Snippet: Additionally we processed three slices each for double staining of EGR1 with dynorphin (goat polyclonal anti-DYN; 1:50, Santa Cruz Biotechnology, sc-46313) to presumably label dopamine D1 receptor expressing medium spiny neurons (D1-MSNs) or the dopamine D2 receptor expressing neurons (mouse polyclonal anti-DRD2, 1:50, Santa Cruz Biotechnology, sc-5303) to label D2-MSNs, choline acetyltransferase (goat polyclonal anti-ChAT, 1:166, Millipore, AB144p) to label cholinergic interneurons; or parvalbumin (goat polyclonal anti-PV, 1:1000, Swant, PVG-214), calretinin (goat polyclonal anti-CR, 1:1000, Swant, CG1) and neuropeptide Y (goat polyclonal anti-NPY, 1:500, Novus, NBP1-46535) to label GABAergic interneurons.

Techniques: Expressing, Fluorescence, Immunohistochemistry, Marker, Microscopy

Journal: Cell reports

Article Title: Thbs1 regulates skeletal muscle mass in a TGFβ-Smad2/3-ATF4-dependent manner

doi: 10.1016/j.celrep.2024.114149

Figure Lengend Snippet:

Article Snippet: Mouse monoclonal anti-TGF beta RII (D-2) , Santa Cruz Biotechnology , Cat# sc-17799; RRID:AB_628348.

Techniques: Virus, Recombinant, Electron Microscopy, Plasmid Preparation, SYBR Green Assay, Membrane, Blocking Assay, Staining, Enzyme-linked Immunosorbent Assay, Activity Assay, Protein Extraction, Cloning, Gene Expression, Software, Microscopy, Real-time Polymerase Chain Reaction