Journal: bioRxiv

Article Title: Distinct features of the Leishmania cap-binding protein LeishIF4E2 revealed by CRISPR-Cas9 mediated heterozygous deletion

doi: 10.1101/2020.05.13.093914

Figure Lengend Snippet: L. amazonensis cells expressing LeishIF4E2-SBP were grown under normal conditions. The cells were washed, fixed in paraformaldehyde and further processed for confocal microscopy. LeishIF4E2 was detected using monoclonal the anti-SBP primary antibody and a secondary goat anti-mouse fluorescent antibody labeled with a green fluorophore (Alexafluore, 488 nM). The nuclear and kinetoplast DNA was stained using DAPI (blue). The images were merged. Images were taken using confocal microscopy showing a Z-projection that was produced by the Image J software. Scale bar: 2µm

Article Snippet: The gels were blotted and subjected to western analysis using monoclonal mouse anti-puromycin antibodies (DSHB, 1:1,000) and secondary peroxidase labeled anti-mouse antibodies (KPL, 1:10,000).

Techniques: Expressing, Confocal Microscopy, Labeling, Staining, Produced, Software

Journal: iScience

Article Title: Naive and memory CD4 + T cell subsets can contribute to the generation of human Tfh cells

doi: 10.1016/j.isci.2021.103566

Figure Lengend Snippet: CyTOF antibody panel

Article Snippet: Vio Bright FITC anti-human CXCR3 , Miltenyi Biotec , Cat# 130-118-673; RRID:AB_2734057.

Techniques:

Journal: iScience

Article Title: Naive and memory CD4 + T cell subsets can contribute to the generation of human Tfh cells

doi: 10.1016/j.isci.2021.103566

Figure Lengend Snippet: Selected genes involved in Tfh cell biology

Article Snippet: Vio Bright FITC anti-human CXCR3 , Miltenyi Biotec , Cat# 130-118-673; RRID:AB_2734057.

Techniques: Activation Assay

Journal: iScience

Article Title: Naive and memory CD4 + T cell subsets can contribute to the generation of human Tfh cells

doi: 10.1016/j.isci.2021.103566

Figure Lengend Snippet: Distinct CD4 + T cell subsets contribute to the generation of Tfh with heterogeneous functional profiles (A) Mean fluorescence intensity of the CXCR5 marker expressed by CXCR5 + PD-1 + cells derived from (1) naive, (2) MemPD-1 neg , (3) MemPD-1 neg and Tfh. (B) Frequency of IL-21- and/or IFNγ-positive cells among CXCR5 + PD-1 + cells at day 3. (C – E) Representative flow plots showing CXCR3, ICOS, and CD40L expression by CXCR5 + PD-1 + cells (left panel) and frequency of CXCR3-, ICOS-, and CD40L-positive cells among CXCR5 + PD-1 + cells at day 3 (right panel). (F) Ex vivo cells or their respective Tfh D3 counterparts obtained after 3 days of splenocyte culture were co-cultured with autologous B cells for 7 days. (G) Box plots represent the frequency of CD27 + CD38 + cells among CD19 + cells, the concentration of total immunoglobulins and the absolute number of live B cells after co-culture. (H) Quantification of IgG1, IgG4, and IgA in the co-culture supernatants. Each symbol (A–H) represents an individual donor. (A–H) A Wilcoxon matched pairs test was performed, ∗, p < 0.05; ∗∗, p < 0.005; ∗∗∗, p < 0.001.

Article Snippet: Vio Bright FITC anti-human CXCR3 , Miltenyi Biotec , Cat# 130-118-673; RRID:AB_2734057.

Techniques: Functional Assay, Fluorescence, Marker, Derivative Assay, Expressing, Ex Vivo, Cell Culture, Concentration Assay, Co-Culture Assay

Journal: iScience

Article Title: Naive and memory CD4 + T cell subsets can contribute to the generation of human Tfh cells

doi: 10.1016/j.isci.2021.103566

Figure Lengend Snippet:

Article Snippet: Vio Bright FITC anti-human CXCR3 , Miltenyi Biotec , Cat# 130-118-673; RRID:AB_2734057.

Techniques: Cell Analysis, Purification, Virus, Recombinant, Blocking Assay, Antibody Labeling, Transfection, Software

Journal: Molecular and cellular endocrinology

Article Title: SDHB and SDHD silenced pheochromocytoma spheroids respond differently to tumour microenvironment and their aggressiveness is inhibited by impairing stroma metabolism.

doi: 10.1016/j.mce.2022.111594

Figure Lengend Snippet: Fig. 1. Characterization of stable SDHB and SDHD silenced cell lines. (A) Representative blots of the expression of mitochondrial SDHA, B and D subunits. (B) Densitometric analysis of western blot bands, performed by Bio-Rad imaging and analysis software (Quantity One), showed significant differences in the SDHB and SDHD subunit expression levels in SDHB (light grey) and in SDHD silenced cells (dark grey) respectively, compared to Wt (black). Bars are the means of three independent preparations ± SD, ***p < 0.001. (C) Representative traces of SDH enzymatic activity measured in cell homogenates. The silenced SDHB and SDHD cells (dotted and continuous lines, respectively) showed a similar decrease of the SDH activity, significantly different compared with Wt (dashed line). (D) Histogram represents the SDH activity expressed as the percentages. SDHB and SDHD silenced cells (light and dark grey, respectively) showed a significantly decreased of SDH activity compared to Wt (black). Bars are the means of three independent experiments (each of them conducted in duplicate samples) ± SD, ***p < 0.01. (E) The bar graph represents the means of intracellular succinate/fumarate ratio ± SD, measured by GC/MS, in three independent experiments with two replicates. SDHB silenced cells (light grey) showed a significant increase of the metabolites ratio compared with both Wt (black) and to SDHD silenced cells (dark grey), *p < 0.05, **p < 0.01. (F) Representative immunoblot of HIF1α expression in Wt, SDHB and SDHD silenced cells. (G) Optical density analysis of western blot bands. Actin im munoblots was used as loading control. For all the analyses One-way ANOVA post-test Bonferroni was used.

Article Snippet: Bound antibodies were detected using ECL reagents (Immobilon) and analysed with a Bio-Rad ChemiDoc Imaging System (Quantity One) for dedicated chemiluminescent image acquisition.

Techniques: Expressing, Western Blot, Imaging, Software, Activity Assay, Gas Chromatography-Mass Spectrometry, Control

Journal: PloS one

Article Title: Kinetic analysis of mouse brain proteome alterations following Chikungunya virus infection before and after appearance of clinical symptoms.

doi: 10.1371/journal.pone.0091397

Figure Lengend Snippet: Figure 5. Western blot validations of differentially regulated proteins identified by 2D-DIGE and/or iTRAQ analyses. (A) Protein samples from each group used for proteomic analysis were minimally labeled with cyanine-3 dye. At the top, a representative protein profile of three biological replicates from brain lysates of mock (M), early (E), late paralytic (LP) and late tetanus-like (LT), separated by 10% SDS-PAGE is shown. WB with fluorescence-based methods was used to detect an overlaid fluorescent scan of the general protein patterns (Cy3 dye; green) and the specific immunoreactive proteins (FITC or Cy5 dye; red). To better visualize protein detection signals observed with each specific antibody used, corresponding cropped WB images are presented in grey levels. (B) The graphs correspond to the mean 6 S.D. of protein quantity measured by densitometry of the antigenic bands. Densitometry analyses were performed using TotalLab Quant v12.2 software (Nonlinear Dynamics), and data were normalized to levels of global protein pattern intensity. The values indicated under each graph correspond to fold changes from paired comparisons. The significance of the differential protein expression are indicated *, p,0.05; **, p,0.01; ***, p,0.001. A.U., arbitrary units. ANXA2: annexin A2; ARRB1: b-arrestin; GABRA1: c-aminobutyric acid receptor subunit alpha-1; GRASP1: GRIP-associated protein; ITGAV: integrin aV; MYPT1: myosin phosphatase target subunit 1; N-Ras: N-Ras; RABEP1: rabaptin-5; SYNGR3: synaptogyrin-3. doi:10.1371/journal.pone.0091397.g005

Article Snippet: Blots were saturated with 5% nonfat dried milk in PBS containing 0.05% (v/v) Tween 20 (PBS-T-milk) for 1 h. Western blot (WB) analyses were carried out with rabbit mono- or polyclonal antibodies directed against b-arrestin (1:5000, ARRB1, no. 4674, Cell Signaling Technology, Danvers, MA), GRIP-associated protein (1:500, GRASP1, no. sc-135681, Santa Cruz Biotechnology, Inc., Santa Cruz, CA), annexin A2 (1:100, ANXA2, no. sc-9061, Santa Cruz), integrin aV (1:100, ITGAV, no. 10179, Santa Cruz), myosin phosphatase target subunit 1 (1:100, MYPT1, no. sc25618, Santa Cruz), rabaptin-5 (1:1000, RABEP1, no. sc-15351, Santa Cruz), N-Ras (1:500, N-Ras, no. sc-519, Santa Cruz), synaptogyrin-3 (1:1000, SYNGR3, no. sc-68936, Santa Cruz), or with a goat polyclonal antibody directed against c-aminobutyric acid receptor subunit alpha-1 (1:100, GABAARa1 or GABRA1, no. sc-31045, Santa Cruz), diluted in PBS-T-milk and incubated overnight at 4uC.

Techniques: Western Blot, Multiplex sample analysis, Labeling, SDS Page, Fluorescence, Software, Expressing

Journal: Molecular Biology Reports

Article Title: Adenovirus-mediated stromal cell-derived factor-1 alpha gene transfer improves cardiac structure and function after experimental myocardial infarction through angiogenic and antifibrotic actions

doi: 10.1007/s11033-009-9642-z

Figure Lengend Snippet: SDF-1 expression and peripheral blood mononuclear cells (PBMNCs) count. a , b Immunofluorescence staining for hSDF-1α expression in the infarction area and non-infarction area 7 day after MI.RT-PCR for SDF-1 mRNA expression in different time point after MI ( c ). ELISA for SDF-1 levels in heart tissues ( d ) and serum ( e ) in the AdV.SDF-1-group. f PBMNCs counts after MI. Values are mean ± SD. ( n = 5, each). * Denotes P = 0.005 vs. control group. a 100×; b 400×

Article Snippet: Western analysis was carried out using the following primary antibodies: transforming growth factor-β1 (TGF-β1), tissue inhibitor of metalloproteinase-1 (TIMP-1), TIMP-2 (1:500, Santa Cruz).

Techniques: Expressing, Immunofluorescence, Staining, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Control

Journal: Molecular Biology Reports

Article Title: Adenovirus-mediated stromal cell-derived factor-1 alpha gene transfer improves cardiac structure and function after experimental myocardial infarction through angiogenic and antifibrotic actions

doi: 10.1007/s11033-009-9642-z

Figure Lengend Snippet: Representative immunofluorescent staining of homing stem cells in the myocardium infarction area. a , b c-Kit staining 7 days after MI. c-Kit positive cells were observed in the infarction area of the AdV.SDF-1 ( a 400×) and control groups ( b 400×). c c-Kit and CXCR4 double-staining 7 days after MI ( c 400×). c-Kit + ( arrow indicated), red fluorescence; cellular nucleus marked by DAPI, blue fluorescence; CXCR4 + ( diamond arrow indicated), green fluorescence. c-Kit + CXCR4 + cells, round arrow indicated. d The number of c-Kit + and CXCR4 + cells were analyzed per high power field (200×) by count method using image pro5.02, respectively. Data are mean ± SD ( n = 5 each). e , f RT-PCR analysis of c-Kit ( e ) and CXCR4 ( f ) mRNA expressions in the infarction area. Rat GAPDH served as an internal control. Data are the means of experiments carried out in duplicate. Values are mean ± SD. ( n = 5, each). * Denotes P = 0.003 vs. control or AdV.LacZ group

Article Snippet: Western analysis was carried out using the following primary antibodies: transforming growth factor-β1 (TGF-β1), tissue inhibitor of metalloproteinase-1 (TIMP-1), TIMP-2 (1:500, Santa Cruz).

Techniques: Staining, Control, Double Staining, Fluorescence, Reverse Transcription Polymerase Chain Reaction

Journal: Molecular Biology Reports

Article Title: Adenovirus-mediated stromal cell-derived factor-1 alpha gene transfer improves cardiac structure and function after experimental myocardial infarction through angiogenic and antifibrotic actions

doi: 10.1007/s11033-009-9642-z

Figure Lengend Snippet: Immunofluorescent staining of myocardium and blood vessel. a – d cTnt staining 28 days after MI. cTnt positive cells were observed in the infarction area of the control ( a ), AdV.LacZ ( b ), AdV.SDF-1 ( c ) and sham groups ( d ). e – h Von Willebrand factor (vWF) staining after MI. blood vessel was observed in the infarction area of the control ( e ), AdV.LacZ ( f ), AdV.SDF-1 ( g ) and sham groups ( h ), respectively. i The number of blood vessel was analyzed per high power field (200×) by count method using image pro5.02, respectively. Data are mean ± SEM ( n = 5 each). j The value of cTnt positive red fluorescence photodensity was analyzed by per high power field (200×) by densitometric method using image pro5.02. Data are mean ± SEM ( n = 5 each). * Denotes P = 0.001 vs. control or AdV.LacZ group

Article Snippet: Western analysis was carried out using the following primary antibodies: transforming growth factor-β1 (TGF-β1), tissue inhibitor of metalloproteinase-1 (TIMP-1), TIMP-2 (1:500, Santa Cruz).

Techniques: Staining, Control, Fluorescence

Journal: Molecular Biology Reports

Article Title: Adenovirus-mediated stromal cell-derived factor-1 alpha gene transfer improves cardiac structure and function after experimental myocardial infarction through angiogenic and antifibrotic actions

doi: 10.1007/s11033-009-9642-z

Figure Lengend Snippet: SYBR Green real-time PCR analysis of TGF-β1 in infarcted ( a ) and noninfarcted ( b ) myocardium, and of collagen type I ( c ), type III ( d ), bFGF (e) and VEGF (f) mRNA expression in infarcted myocardium. Data were processed with a specially designed software program based on Ct values of each sample and normalized to GAPDH. Bars represent mRNA levels relative to GAPDH mRNA level for control, AdV.LacZ, AdV.SDF-1 and sham group. Data are the means of experiments carried out in duplicate. Values are mean ± SD. ( n = 5 each). * P = 0.002 vs. control or AdV.LacZ group. VEGF vascular endothelial growth factor, basic-FGF basic fibroblast growth factor

Article Snippet: Western analysis was carried out using the following primary antibodies: transforming growth factor-β1 (TGF-β1), tissue inhibitor of metalloproteinase-1 (TIMP-1), TIMP-2 (1:500, Santa Cruz).

Techniques: SYBR Green Assay, Real-time Polymerase Chain Reaction, Expressing, Software, Control

Journal: Molecular Biology Reports

Article Title: Adenovirus-mediated stromal cell-derived factor-1 alpha gene transfer improves cardiac structure and function after experimental myocardial infarction through angiogenic and antifibrotic actions

doi: 10.1007/s11033-009-9642-z

Figure Lengend Snippet: Western blotting analysis of TGF-β 1 , TIMP-1 and TIMP-2 in infarcted myocardium. Bars represent protein levels relative to β-tubin for control group, sham group and AdV-SDF-1 group. Data are the means of experiments carried out in duplicate. Values are mean ± SD. * Denotes P = 0.004 vs. sham or control group ( n = 5 each)

Article Snippet: Western analysis was carried out using the following primary antibodies: transforming growth factor-β1 (TGF-β1), tissue inhibitor of metalloproteinase-1 (TIMP-1), TIMP-2 (1:500, Santa Cruz).

Techniques: Western Blot, Control

Journal: Molecular Biology Reports

Article Title: Adenovirus-mediated stromal cell-derived factor-1 alpha gene transfer improves cardiac structure and function after experimental myocardial infarction through angiogenic and antifibrotic actions

doi: 10.1007/s11033-009-9642-z

Figure Lengend Snippet: Effects of SDF-1 on histology and form. a – d Representative picture of a myocardial infarction 28 days after surgery, the heart was exercised, and infarction size was measured by TTC (3×). e – h The infarcted myocardium was unstained by TTC ( white , arrow indicated), and the non-ischemic zones stained brick red. i – p Representative Masson’s trichrome-stained sections in the area of infarcted left ventricular tissue 28 days after MI ( blue , collagen , black arrow indicated; i – l 3×; m – p 400×). There was a decreased infarct size, thicker LV wall in the AdV.SDF-1 group. The new or survivaling myocardium ( black round arrow indicated) typically appeared as red-positive cells wedged between an inner and outer layer of collagen (each group, n = 6) (Color figure online)

Article Snippet: Western analysis was carried out using the following primary antibodies: transforming growth factor-β1 (TGF-β1), tissue inhibitor of metalloproteinase-1 (TIMP-1), TIMP-2 (1:500, Santa Cruz).

Techniques: Staining

Journal: Molecular Biology Reports

Article Title: Adenovirus-mediated stromal cell-derived factor-1 alpha gene transfer improves cardiac structure and function after experimental myocardial infarction through angiogenic and antifibrotic actions

doi: 10.1007/s11033-009-9642-z

Figure Lengend Snippet: Effects of SDF-1 on infarct size and left ventricle wall thickness. a Quantitation of collagen content in percent of infarct and noninfarcted area in multiple fields. b Thickness of infarct LV wall in AdV.SDF-1 group is obviously thicker than the infarct LV wall in the control and AdV.Lac-Z group. Thickness of infarct LV wall in AdV.SDF-1 group is thinner than the non-infarct LV wall ( & P = 0.001) and is obviously thicker than the infarct LV wall in the control or AdV.Lac-Z group ( & P = 0.0005). The average LV wall in the AdV.SDF-1 group is thicker than in the control or AdV.Lac-Z group ( # P = 0.001) and thinner than in the sham group ( # P = 0.0003). The non-infarct LV wall in the AdV.SDF-1 group is thinner than in the sham group (* P = 0.002) and thicker than in the control or AdV.Lac-Z group ( * P = 0.0003). c There were statistically significant differences in infarct size among the control, AdV.Lac-Z and AdV.SDF-1 groups (mean ± SD). (* P = 0.0006). d Expansion index analyses in AdV.SDF-1 group showed lower than control or AdV.Lac-Z group (each group, n = 6)

Article Snippet: Western analysis was carried out using the following primary antibodies: transforming growth factor-β1 (TGF-β1), tissue inhibitor of metalloproteinase-1 (TIMP-1), TIMP-2 (1:500, Santa Cruz).

Techniques: Quantitation Assay, Control

Journal: Molecular Biology of the Cell

Article Title: The ubiquitin ligase deltex-3l regulates endosomal sorting of the G protein–coupled receptor CXCR4

doi: 10.1091/mbc.e13-10-0612

Figure Lengend Snippet: FIGURE 1: A role for DTX3L in CXCR4 degradation. (A) HeLa cells transfected with siRNA directed against control, AIP4, or DTX3L were treated with vehicle (PBS + 0.1% BSA) or 10 nM CXCL12 for 3 h. Whole-cell lysates were analyzed for the levels of endogenous CXCR4 and the indicated proteins by immunoblotting (IB). (B) CXCR4 levels normalized to actin were determined by densitometric analysis. Data represent the average percentage CXCR4 degraded in CXCL12-treated vs. vehicle-treated cells. Representative immunoblots are shown in A, and B represents quantification of eight independent experiments. Error bars represent SEM. Data were analyzed by a one-way ANOVA (p < 0.0001), followed by Bonferroni’s posthoc test. CXCR4 degradation in AIP4 (p < 0.001) and DTX3L (p < 0.001) siRNA–treated cells was significantly different from siRNA control (siCtrl).

Article Snippet: The mouse monoclonal antibodies directed against HRS (c-19) and ubiquitin (P4D1) and the goat polyclonal directed against DTX3L (N-16) were from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Transfection, Control, Western Blot

Journal: Molecular Biology of the Cell

Article Title: The ubiquitin ligase deltex-3l regulates endosomal sorting of the G protein–coupled receptor CXCR4

doi: 10.1091/mbc.e13-10-0612

Figure Lengend Snippet: FIGURE 2: DTX3L localizes to early endosomes upon CXCR4 activation. (A) HeLa cells were treated with vehicle or CXCL12 for 30 min. Cells were then fixed, permeabilized, and incubated with antibodies directed against DTX3L and the early endosomal marker EEA1 or the lysosomal marker LAMP2. Inset represents 3× enlargement of the boxed region. Yellow in the merged image indicates colocalization between DTX3L and EEA1 or LAMP2. Differential interference contrast (DIC) images are shown. Equal acquisition settings (gain and intensity) were used between parallel samples within each experiment. Representative images from four independent experiments. (B) CXCR4 activation increases DTX3L puncta number in HeLa cells. DTX3L puncta were counted using ImageJ software. Data represent the average puncta count from 35–45 cells from four independent experiments. Error bars represent SEM (not visible). (C, D) Pearson product–moment correlation coefficient calculated to determine the level of colocalization between DTX3L and EEA1 (C) and LAMP2 (D). Data were analyzed by Student’s t test.

Article Snippet: The mouse monoclonal antibodies directed against HRS (c-19) and ubiquitin (P4D1) and the goat polyclonal directed against DTX3L (N-16) were from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Activation Assay, Incubation, Marker, Software

Journal: Molecular Biology of the Cell

Article Title: The ubiquitin ligase deltex-3l regulates endosomal sorting of the G protein–coupled receptor CXCR4

doi: 10.1091/mbc.e13-10-0612

Figure Lengend Snippet: FIGURE 3: DTX3L regulates CXCR4 sorting from early endosomes to lysosomes. (A) HeLa cells transfected with siRNA directed against control or DTX3L were treated with vehicle (PBS + 0.1% BSA) or 10 nM CXCL12 for 3 h. Cells were then fixed, permeabilized, and incubated with antibodies directed against CXCR4, DTX3L, early endosomal marker EEA1, or late endosomal/lysosomal marker LAMP2. Inset represents 3× enlargement of the boxed region. Yellow in the merged image indicates colocalization between CXCR4 and EEA1 or LAMP2. DIC images are shown. Equal acquisition settings (gain and intensity) were used between parallel samples within each experiment. Representative images are from four independent experiments. (B) Pearson product–moment correlation coefficient determined using the ImageJ plug-in Colocalization Finder. Data represent the average from four independent experiments. Data were analyzed by a one-way ANOVA (p < 0.001), followed by Bonferroni’s posthoc test. (C) CXCR4 puncta counted using the ImageJ function Analyze Particles. Data represent the average puncta count from 45–60 cells from three independent experiments. Error bars represent SEM. Data were analyzed by Student’s t test.

Article Snippet: The mouse monoclonal antibodies directed against HRS (c-19) and ubiquitin (P4D1) and the goat polyclonal directed against DTX3L (N-16) were from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Transfection, Control, Incubation, Marker

Journal: Molecular Biology of the Cell

Article Title: The ubiquitin ligase deltex-3l regulates endosomal sorting of the G protein–coupled receptor CXCR4

doi: 10.1091/mbc.e13-10-0612

Figure Lengend Snippet: FIGURE 4: AIP4 and DTX3L interact directly and form a complex in HeLa cells. (A) Cleared lysates from HeLa cells (500 μg) were incubated with either anti-IgG control or anti-DTX3L antibodies to immunoprecipitate endogenous DTX3L. (B) Equimolar amounts of His-tagged DTX3L (1–10 nM) were incubated with equal amounts of purified GST-AIP4 (100 nmol). Samples were resolved by 10% SDS–PAGE and analyzed by immunoblotting for DTX3L or GST. Input represents 10% of HIS-DTX3L used in the binding reaction. Data represent the fold change in GST-AIP4 binding to HIS-DTX3L. Data are representative of three independent experiments. Data were analyzed by one-way ANOVA, followed by Bonferroni’s posthoc test. Data show a significant increase in His-DTX3L binding to GST-AIP4 with increasing His-DTX3L concentration. (C) HeLa lysates (500 μg) were treated with CXCL12 (10 nM) and incubated with either anti-IgG control or anti-DTX3L antibodies to immunoprecipitate endogenous DTX3L. Samples were resolved by 10% SDS–PAGE and analyzed by immunoblotting with the indicated antibodies. The amount of AIP4 binding was quantitated by densitometric analysis from four independent experiments and then analyzed by one-way ANOVA, followed by followed by Bonferroni’s posthoc test for significance (p < 0.05). Error bars represent the SEM. (D) HeLa cells transfected with FLAG-tagged AIP4 were treated with 10 nM CXCL12 for 0–60 min. Cells were fixed, permeabilized, and incubated with antibodies directed against FLAG-AIP4, DTX3L, and EEA1. Representative images are from three independent experiments. DIC images are shown. Inset, 3× enlargement of the boxed region. Equal acquisition settings (gain and intensity) were used between parallel samples within each experiment. (E) Immunoblot showing level of FLAG-AIP4 expression over endogenous AIP4. (F) FLAG-AIP4 and DTX3L show strong colocalization, as determined by calculating the Pearson product–moment correlation coefficient. (G, H) Puncta were counted using the particle analysis software of ImageJ. Data represent the average FLAG-AIP4 (G) or DTX3L (H) puncta per cell from four independent experiments. Data were analyzed by one-way ANOVA, followed by Bonferroni’s posthoc test.

Article Snippet: The mouse monoclonal antibodies directed against HRS (c-19) and ubiquitin (P4D1) and the goat polyclonal directed against DTX3L (N-16) were from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Incubation, Control, Purification, SDS Page, Western Blot, Binding Assay, Concentration Assay, Transfection, Expressing, Particle Size Analysis, Software

Journal: Molecular Biology of the Cell

Article Title: The ubiquitin ligase deltex-3l regulates endosomal sorting of the G protein–coupled receptor CXCR4

doi: 10.1091/mbc.e13-10-0612

Figure Lengend Snippet: FIGURE 6: DTX3L regulates the distribution of HRS and STAM1. (A, D) HeLa cells transfected with either control or DTX3L siRNA were treated with 10 nM CXCL12 for 30 min. Cells were fixed, permeabilized, and incubated with antibodies directed against DTX3L, HRS, (A) and STAM-1 (D). Inset, 3× enlargement of the boxed region. Yellow in the merged image indicates colocalization between HRS (A) or STAM-1 (D) and DTX3L. DIC images are shown. Equal acquisition settings (gain and intensity) were used between parallel samples within each experiment. (B, E) HRS and STAM-1 puncta are reduced in DTX3L siRNA–treated cells. Puncta were counted using the particle analysis software of ImageJ. Data represent the average HRS (B) or STAM-1 (E) puncta per cell from four and three independent experiments, respectively. For the HRS puncta analysis, 110–145 cells were examined, and for the STAM-1 puncta analysis, 45–50 cells were examined. Data were analyzed by Student’s t test. DTX3L shows strong colocalization with HRS (C) and STAM-1 (F) in control siRNA–treated cells, as determined by calculating the Pearson product–moment correlation coefficient.

Article Snippet: The mouse monoclonal antibodies directed against HRS (c-19) and ubiquitin (P4D1) and the goat polyclonal directed against DTX3L (N-16) were from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Transfection, Control, Incubation, Particle Size Analysis, Software

Journal: Molecular Biology of the Cell

Article Title: The ubiquitin ligase deltex-3l regulates endosomal sorting of the G protein–coupled receptor CXCR4

doi: 10.1091/mbc.e13-10-0612

Figure Lengend Snippet: FIGURE 7: DTX3L inhibits the E3 ubiquitin ligase activity of AIP4. (A) Purified AIP4, AIP4-C830A, HIS-DTX3L, or HIS-DTX3L-RING domain mutant (3C/A) were incubated with ATP, E1, E2 (UbcH5c), and ubiquitin in a final volume of 20 μl at room temperature for 1½ h. Reactions were stopped by the addition of 20 μl of 2× sample buffer. Proteins were resolved by 7% SDS–PAGE and analyzed by immunoblotting with the indicated antibodies. The C830A cleavage from GST was not complete, resulting in detection of multiple bands in both the input and reaction lanes (indicated with asterisks) that correspond to uncleaved and degraded products of GST-C830A, as determined by immunoblot analysis with an anti-GST antibody (unpublished data). A high–molecular weight nonspecific band is also noted with an asterisk. Data are representative of six independent experiments. (B) Purified AIP4, HIS-DTX3L, or MBP-Parkin were incubated with ATP, E1, E2 (UbcH5c), and ubiquitin in a final volume of 20 μl at room temperature for 1½ h. Reactions were stopped by the addition of 20 μl of 2× sample buffer. Proteins were resolved by 7% SDS–PAGE and analyzed by immunoblotting with the indicated antibodies. Data are representative of four independent experiments. (C) Hyperubiquitination of HRS in DTX3L-depleted cells is suppressed by AIP4 depletion. HeLa cells were transfected with siRNA directed against control, DTX3L, or AIP4 plus FLAG-ubiquitin and T7-HRS. Samples were processed as described in the legend to Figure 5, A and B. Data are representative of four independent experiments.

Article Snippet: The mouse monoclonal antibodies directed against HRS (c-19) and ubiquitin (P4D1) and the goat polyclonal directed against DTX3L (N-16) were from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Ubiquitin Proteomics, Activity Assay, Purification, Mutagenesis, Incubation, SDS Page, Western Blot, High Molecular Weight, Transfection, Control

Journal: iScience

Article Title: Methods to separate nuclear soluble fractions reflecting localizations in living cells

doi: 10.1016/j.isci.2021.103503

Figure Lengend Snippet: Key resources table

Article Snippet: The antibodies used in this study were as follows: mAb414 ( Davis and Blobel, 1986 ) (902902; BioLegend) at 1:500 for IF, QE5 ( Panté et al., 1994 ) (ab24700; Abcam) at 1:100 for IF, RL2 ( Holt et al., 1987 ; Snow et al., 1987 ) (NB300-524; Novus biologicals) at 1:200 for IF, mouse anti-GAPDH (sc-32233; Santa Cruz Biotechnology) at 1:100 for IF and 1:1000 for WB, mouse anti-α-tubulin (T9026; Merck) at 1:500 for IF and 1:1000 for WB, goat anti-RanBP1 (ab17034; Abcam) at 1:200 for IF and 1:1000 for WB, mouse anti-Lamin A/C (sc-7292; Santa Cruz Biotechnology) at 1:200 for both IF and WB, rabbit anti-Histone H3 (ab1791; Abcam) at 1:200 for IF and 1:5000 for WB, goat anti-RCC1 (sc-1162; Santa Cruz Biotechnology) at 1:200 for both IF and WB, mouse anti-PCNA (610665; BD Biosciences) at 1:2000 for WB, mouse anti-PCNA (sc-56; Santa Cruz Biotechnology) at 1:50 for IF, rabbit anti-RanBP3 (NB100-74647; Novus biologicals) at 1:100 for IF and 1:1000 for WB, mouse anti- profilin 1 (PFN1) (67390-1-Ig; Proteintech) at 1:100 for IF and 1:2000 for WB, rabbit anti- thioredoxin (TXN) (14999-1-AP; Proteintech) at 1:50 for IF and 1:1000 for WB, rabbit anti-Calnexin (2679; Cell Signaling TECHNOLOGY) at 1:1000 for WB, rabbit anti-PDI (3501; Cell Signaling TECHNOLOGY) at 1:1000 for WB, rabbit anti-RCAS1 (12290; Cell Signaling TECHNOLOGY) at 1:1000 for WB, rabbit anti-HINT1 (10717-1-AP; Proteintech) at 1:100 for IF and 1:1000 for WB, mouse anti-ACP1 (sc-390190; Santa Cruz Biotechnology) at 1:100 for IF and 1:1000 for WB, mouse anti-BLVRB (sc-373692; Santa Cruz Biotechnology) at 1:50 for IF and 1:1000 for WB, and mouse anti-Ran (610341; BD Biosciences) at 1:500 for IF and 1:1000 for WB.

Techniques: Recombinant, Protease Inhibitor, Plasmid Preparation, Magnetic Beads, Bicinchoninic Acid Protein Assay, Multiplex sample analysis, Multiplex Assay, Extraction, Software, Microscopy

Journal: eLife

Article Title: METTL18-mediated histidine methylation of RPL3 modulates translation elongation for proteostasis maintenance

doi: 10.7554/eLife.72780

Figure Lengend Snippet:

Article Snippet: Antibody , Anti-RPL3 (mouse monoclonal) , Proteintech Group , Cat# 66130-1-lg; RRID :AB_2881529 , WB (1:1000).

Techniques: CRISPR, Expressing, Transfection, Construct, RNA Expression, Control, Recombinant, Sequencing, Mutagenesis, Reporter Assay, Software, Methylation, Multiplex sample analysis

Journal: iScience

Article Title: Naive and memory CD4 + T cell subsets can contribute to the generation of human Tfh cells

doi: 10.1016/j.isci.2021.103566

Figure Lengend Snippet: CyTOF antibody panel

Article Snippet: Pure anti-human CCR5 , Miltenyi Biotec , Cat# 130-122-313; RRID:AB_2801894.

Techniques:

Journal: iScience

Article Title: Naive and memory CD4 + T cell subsets can contribute to the generation of human Tfh cells

doi: 10.1016/j.isci.2021.103566

Figure Lengend Snippet: Selected genes involved in Tfh cell biology

Article Snippet: Pure anti-human CCR5 , Miltenyi Biotec , Cat# 130-122-313; RRID:AB_2801894.

Techniques: Activation Assay

Journal: iScience

Article Title: Naive and memory CD4 + T cell subsets can contribute to the generation of human Tfh cells

doi: 10.1016/j.isci.2021.103566

Figure Lengend Snippet:

Article Snippet: Pure anti-human CCR5 , Miltenyi Biotec , Cat# 130-122-313; RRID:AB_2801894.

Techniques: Cell Analysis, Purification, Virus, Recombinant, Blocking Assay, Antibody Labeling, Transfection, Software