Journal: Experimental & Molecular Medicine

Article Title: The Mycobacterium avium subsp. paratuberculosis fibronectin attachment protein, a toll-like receptor 4 agonist, enhances dendritic cell-based cancer vaccine potency

doi: 10.3858/emm.2012.44.5.038

Figure Lengend Snippet: FAP induces the expression of MHC class I and II and co-stimulatory molecules (CD80 and CD 86) in wild type and TLR2 -/- BMDCs, but the expression of those molecules was impaired in TLR4 -/- BMDCs. On day 6 of culturing, BMDCs were pretreated with or without the indicated GSK-3 inhibitor (SB415286) concentration for 30 min and then harvested after 24 h of incubation with FAP (500 ng/ml). BMDCs were first gated for CD11c + cells and analyzed by two-color flow cytometry. (A) FAP induces the expression of co-stimulatory molecules (CD80 and CD86) in wild type and TLR2 -/- BMDCs, but the expression of these molecules was impaired in TLR4 -/- BMDCs. The mean fluorescence intensity (MFI) of double-positive cells is shown for each panel. The mean ± SEM values shown represent 3 independent experiments. ** P < 0.01 compared with untreated BMDCs. (B) FAP induces expression of MHC class I and II in wild type and TLR2 -/- BMDCs, but the expression of these molecules was impaired in TLR4 -/- BMDCs. The mean fluorescence intensity (MFI) of double-positive cells is shown for each panel. The mean ± SEM values shown represent 3 independent experiments. * P < 0.05 compared with untreated BMDCs.

Article Snippet: Fluorescein isothiocyanate (FITC)- or phycoerythrin (PE)-conjugated monoclonal antibodies (mAbs) used to detect the expression of TLR4, CD11c (HL3), CD80 (16-10A1), CD86 (GL1), I-A b β-chain (AF-120.1), and H-2K b (AF6-88.5) by flow cytometry, as well as isotype-matched control mAbs and the biotinylated anti-CD11c (N418) mAb were purchased from R&D Systems.

Techniques: Expressing, Concentration Assay, Incubation, Flow Cytometry, Fluorescence

Journal: Journal of Alzheimer's Disease

Article Title: Changes in the Expression of Genes Associated with Intraneuronal Amyloid-β and Tau in Alzheimer's Disease

doi: 10.3233/jad-2010-1216

Figure Lengend Snippet: Fig. 1. LCM of Aβ-associated and non-associated neurons from AD hippocampal tissue section. Left panel, immunostaining using FITC-conjugated Aβ antibody. Middle panel, Nissl stain of neurons (dark areas). Right panel, laser capture of Nissl-stained neurons that overlap with Aβ immunostaining were microdissected and collected as Aβ-associated neurons. Nissl-stained neurons without Aβ immunostaining were collected as Aβ-free neurons.

Article Snippet: After incubation with antibodies to either Aβ or P-tau, antibody binding was detected with biotinylated antirabbit or mouse IgG (1/200 dilution; 20 min at room temperature; Vector Laboratories, Burlingame, CA), a 1:300 dilution of FITC-avidin DCS (Vector Laboratories) for 5 min, followed by avidin/biotin blocking for 15 min, and then immunostaining with BDNF, TrkB, or DYN and detection with biotinylated anti-rabbit or mouse IgG and Texas Red DCS for 5 min. Pseudocolor images were captured using a Nikon fluorescence microscope with FITC and Texas Red filters using IP Lab software (Fairfax, VA) and merged using Adobe Photoshop (San Jose, CA).

Techniques: Immunostaining, Staining

Journal: Journal of Alzheimer's Disease

Article Title: Changes in the Expression of Genes Associated with Intraneuronal Amyloid-β and Tau in Alzheimer's Disease

doi: 10.3233/jad-2010-1216

Figure Lengend Snippet: Fig. 4. Hippocampal cells coexpressing Aβ and TrkB are present in AD and control cases. Representative DIF of Aβ/TrkB showing high levels of Aβ (green) in AD (AD5) compared to control (C5); higher levels of TrkB (red) in control compared to AD; and similar numbers of Aβ/TrkB (yellow cells in merge) in controls compared to AD (x200). Arrows point to examples of coexpressing cells. The bottom two panels are the negative controls using nonspecific mouse or rabbit IgG as primary antibodies and the appropriate FITC-avidin or Texas Red DCS. No immunostaining was detected.

Article Snippet: After incubation with antibodies to either Aβ or P-tau, antibody binding was detected with biotinylated antirabbit or mouse IgG (1/200 dilution; 20 min at room temperature; Vector Laboratories, Burlingame, CA), a 1:300 dilution of FITC-avidin DCS (Vector Laboratories) for 5 min, followed by avidin/biotin blocking for 15 min, and then immunostaining with BDNF, TrkB, or DYN and detection with biotinylated anti-rabbit or mouse IgG and Texas Red DCS for 5 min. Pseudocolor images were captured using a Nikon fluorescence microscope with FITC and Texas Red filters using IP Lab software (Fairfax, VA) and merged using Adobe Photoshop (San Jose, CA).

Techniques: Control, Avidin-Biotin Assay, Immunostaining

Journal: Frontiers in Endocrinology

Article Title: Cyclin D1 Stability Is Partly Controlled by O -GlcNAcylation

doi: 10.3389/fendo.2019.00106

Figure Lengend Snippet: Perturbation of O -GlcNAc cycling affects cyclin D1 protein expression level. MCF7 cells were transfected for 72 h with siRNA (Control (Ctrl), OGT, or OGA) and harvested to get protein lysates or mRNA samples. (A) Western-blotting analysis following siRNA show depletion of targeted proteins and O -GlcNAcylated proteins with concomitant variation in cyclin D1 (cycD1) level. (B) OGT and cycD1 mRNA levels were quantified by qPCR using RPLP0 as internal control. Results correspond to the mean value ± s.d. of three independent experiments (*** p < 0.005). (C) HEK293T cells were seeded in 12-well plates with siRNA (Ctrl, OGT, or OGA) for 24 h and then transfected with pcDNA3.1 or CycD1-FLAG (100 ng). Cells were lysed 2 days later (three independent experiments). Lysate from non-transfected HEK293T cells (n.tf.) was also loaded on the same gel. (D) HEK293T cells were transfected in 12-well plates for 48 h with CycD1-FLAG (500 ng) and OGT-HA (500 ng or 1 μg) and then lysed in Laemmli buffer (two independent experiments). (C,D) The cellular lysates were analyzed by Western blot using specific antibodies. Histograms represent the relative intensity of cyclin D1 expression levels normalized to GAPDH levels. Statistical analyses were performed by Student's t -test ( *** p < 0.005, ** p < 0.05).

Article Snippet: Antibodies against cyclin D1 (DCS-6, sc-20044; A12, sc-8396), HA-tag (sc-805), GAPDH (sc-47724), beta-actin (sc-1616) and normal rabbit or mouse IgG were from Santa Cruz (Heidelberg, Germany).

Techniques: Expressing, Transfection, Control, Western Blot

Journal: Frontiers in Endocrinology

Article Title: Cyclin D1 Stability Is Partly Controlled by O -GlcNAcylation

doi: 10.3389/fendo.2019.00106

Figure Lengend Snippet: Turnover of cyclin D1 is dependent to O- GlcNAc homeostasis. (A–C) MCF7 cells were treated overnight with Ac-5S-G (100 μM) or ThG (1 μM). Then CHX (50 μg/ml) was added into the medium before cell lysis at indicated times. (A) , Western-blots show the levels of O -GlcNAcylated proteins and cyclin D1. GAPDH served as a control for equal loading. (B) The histogram represents the relative quantification of O -GlcNAc levels in treated cells compared with control cells. (C) Graph shows the ratios of cyclin D1 to GAPDH. Representative results of three independent experiments. (D–F) HCT116 cells were pretreated 2 h with vehicle (DMSO, Control), ThG (1 μM), or Ac-5S-G (50 μM). Cells were starved in serum-free medium for the indicated times prior to cell lysis. (D) Western-blot show the levels of O -GlcNAcylated proteins and cyclin D1. Actin served as a control for equal loading. (E) The histogram represents the relative quantification of O -GlcNAc levels in treated cells compared with control cells. (F) The histogram represents the relative intensity of cyclin D1 levels normalized to actin levels (100% is the normalized level of cyclin D1 in the control cells at T0). On each histogram, the percentage of the decrease of cyclin D1 level after 4 h of serum starvation is indicated. Results are representative of two independent experiments. Statistical analysis in ( B , E) was performed by Student's t -test ( *** p < 0.005).

Article Snippet: Antibodies against cyclin D1 (DCS-6, sc-20044; A12, sc-8396), HA-tag (sc-805), GAPDH (sc-47724), beta-actin (sc-1616) and normal rabbit or mouse IgG were from Santa Cruz (Heidelberg, Germany).

Techniques: Lysis, Western Blot, Control, Quantitative Proteomics

Journal: Frontiers in Endocrinology

Article Title: Cyclin D1 Stability Is Partly Controlled by O -GlcNAcylation

doi: 10.3389/fendo.2019.00106

Figure Lengend Snippet: OGT interacts with cyclin D1. (A) HEK293T cells were co-transfected with HA-OGT and Myc-cyclin D1 plasmids for 48 h before cell lysis. Cyclin D1 and OGT were immunoprecipitated and Western-blot analysis were performed to detect OGT, OGA and cyclin D1. Negative controls of IP were done using non-relevant IgG (IgG). (B) HCT116 cells were treated overnight with DMSO (-) or ThG (1 μM) (+) before cell lysis in RIPA buffer and immunoprecipitation of endogenous cyclin D1 and OGT. Western-blot analysis of immunoprecipitates (IP) and Input (Inp.) using OGT and cycD1 antibodies. (C – E) Quiescent MCF7 cells (0) were stimulated with serum for the indicated times. (C) Cell cycle profile was monitored by flow cytometry after staining with propidium iodide. The percentage of cells in each phase is reported, according to serum release for each time point. (D) Cyclin D1 and OGT were detected in synchronized MCF7 cells by confocal indirect immunofluorescence using specific antibodies. Nuclei were counterstained with DAPI. Scale bar, 20 μM. (E) The interaction between OGT and cyclin D1 was detected using in situ PLA and immunofluorescent confocal microscopy. Nuclei were stained with DAPI. Pictures are the merge of PLA signal (AlexaFluo488) and DAPI channels. Quantification of PLA is presented as scatter dot plot; each dot represents the mean of PLA fluorescence intensity in the nucleus of a single cell. Bars represents the median with interquartile range for each experience (one-way ANOVA test, *** p < 0.0001, ** p < 0.005, * p < 0.05). Scale bar, 20 μM.

Article Snippet: Antibodies against cyclin D1 (DCS-6, sc-20044; A12, sc-8396), HA-tag (sc-805), GAPDH (sc-47724), beta-actin (sc-1616) and normal rabbit or mouse IgG were from Santa Cruz (Heidelberg, Germany).

Techniques: Transfection, Lysis, Immunoprecipitation, Western Blot, Flow Cytometry, Staining, Immunofluorescence, In Situ, Confocal Microscopy, Fluorescence

Journal: Frontiers in Endocrinology

Article Title: Cyclin D1 Stability Is Partly Controlled by O -GlcNAcylation

doi: 10.3389/fendo.2019.00106

Figure Lengend Snippet: Cyclin D1 is O -GlcNAcylated. (A) in situ PLA experiments by using anti-cyclin D1 and anti-O-GlcNAc (RL2) antibodies on asynchronous MCF7 cells. Nuclei were stained with DAPI. PLA signal was detected by immunofluorescent confocal microscopy. Scale bar, 20 μM. (B) MCF7 cells were treated with MG132 (4 μM) for 2 h prior cell lysis. Cyc D1-FLAG transfected HEK293T cells were treated overnight or not with ThG (1 μM) and lysed in RIPA buffer. O -GlcNAcylated proteins were enriched on sWGA-agarose beads. Incubation of sWGA beads with 0.5 M GlcNAc was done as a negative control. sWGA-bound Cyclin D1 was detected by Western-blot after lectin affinity chromatography (Input, 30 μg; NR., not retained, 30 μg). (C) Enrichment of biotinylated proteins from CycD1-FLAG transfected HEK293T cells or purified α-crystallin after enzymatic labeling with UDP-GalNAz and click chemistry reaction using a biotin-alkyne probe. Cyclin D1 and α-crystallin were revealed by Western-Blot on avidin-bound protein fraction and Input.

Article Snippet: Antibodies against cyclin D1 (DCS-6, sc-20044; A12, sc-8396), HA-tag (sc-805), GAPDH (sc-47724), beta-actin (sc-1616) and normal rabbit or mouse IgG were from Santa Cruz (Heidelberg, Germany).

Techniques: In Situ, Staining, Confocal Microscopy, Lysis, Transfection, Incubation, Negative Control, Western Blot, Affinity Chromatography, Purification, Labeling, Avidin-Biotin Assay

Journal: Frontiers in Endocrinology

Article Title: Cyclin D1 Stability Is Partly Controlled by O -GlcNAcylation

doi: 10.3389/fendo.2019.00106

Figure Lengend Snippet: Elevation of O -GlcNAc levels decreases ubiquitination of cyclin D1. HEK 293T cells were transfected for 48 h with CycD1-FLAG and Ub-HA plasmids and treated or not with ThG (1 μM) overnight. MG132 (20 μM) was then added into the medium for the indicated times before cell lysis. (A) Input (30 μg) were revealed using anti-HA (Ub) and anti-FLAG antibodies. GAPDH was used as a loading control. (B) The relative ratios of ubiquitin to GAPDH levels were calculated by densitometry using Image J software (mean ± s.d., two independent experiments). (C) CycD1 was immunoprecipitated and Western-blot was revealed using anti-HA (Ub) and anti-CycD1 antibodies. (D) The ratios of the ubiquitinated cycD1 levels were calculated by densitometry using Image J® software (mean ± s.d., three independent experiments). Statistical analysis was performed by Student's t -test (** p < 0.05, *** p < 0.005).

Article Snippet: Antibodies against cyclin D1 (DCS-6, sc-20044; A12, sc-8396), HA-tag (sc-805), GAPDH (sc-47724), beta-actin (sc-1616) and normal rabbit or mouse IgG were from Santa Cruz (Heidelberg, Germany).

Techniques: Ubiquitin Proteomics, Transfection, Lysis, Control, Software, Immunoprecipitation, Western Blot