Journal: The Journal of Biological Chemistry

Article Title: The extreme C-terminal region of kindlin-2 is critical to its regulation of integrin activation

doi: 10.1074/jbc.M117.776195

Figure Lengend Snippet: Conserved residues in the C-terminal region of kindlin-2 support αIIbβ3 activation in αIIbβ3-CHO cells. A, alignment of C-terminal sequences of human K2 and Fit1, the K2 homolog of D. melanogaster. B and C, αIIbβ3-CHO cells were transiently transfected with plasmids encoding EGFP-fused FL, wild-type K2 or its mutants and DsRed talin-H, and activation of αIIbβ3 integrin was determined at 24 h by flow cytometry by staining the cells with the activation-specific antibody PAC-1 (see “Experimental procedures”). Error bars, S.D. Intermediate flow cytometry data (dot plots and histograms) are shown in supplemental Fig. S1.

Article Snippet: The following primary antibodies were used: PAC1, which reacts with the activated but not the resting conformation of integrin αIIbβ3 ( 51 ) (BD Biosciences); 9EG7, which reacts selectively with the activated conformer of β1 integrins ( 41 ); mouse anti-EGFP (Takara Clontech, Mountain View, CA); mouse anti-GFP (clone B2) (Santa Cruz Biotechnology, Inc., Dallas, TX); mouse anti-β1 integrin (BD Transduction Laboratories); mouse anti-human CD41b (BD Biosciences); mouse anti-PSGL-1; clone KPL-1 (EMD Millipore, Temecula, CA); rabbit anti-integrin β3 integrin (Cell Signaling); mouse anti-GST (EMD Millipore); rabbit anti-PSGL-1(Santa Cruz Biotechnology); rabbit anti-kindlin 2 (Cell Signaling); rabbit anti-actin (Cell Signaling); and rabbit anti-ILK (Cell Signaling).

Techniques: Activation Assay, Transfection, Flow Cytometry, Staining

Journal: The Journal of Biological Chemistry

Article Title: The extreme C-terminal region of kindlin-2 is critical to its regulation of integrin activation

doi: 10.1074/jbc.M117.776195

Figure Lengend Snippet: The kindlin-2 C-terminal segment supports integrin function in HEL megakaryotic and RAW 264.7 macrophage-like cells. A, HEL cells were transiently transfected with plasmids encoding EGFP-fused K2 or its mutants and DsRed talin-H, and the extent of αIIbβ3 activation in DsRed and EGFP double-positive cells was quantified by flow cytometry with the activation-specific antibody PAC-1 (see “Experimental procedures”). The experiments were performed three times. Error bars, S.D. The total αIIbβ3 expression, measured with an mAb unaffected by the activation status of the receptor, in the presence and absence of the various K2s with or without talin-H was unaffected. The expression levels of the K2 mutants were similar. B, HEL cells were transiently transfected with plasmids encoding EGFP alone and EGFP-K2 constructs. After 24 h, EGFP expression levels in HEL cells were determined by flow cytometry; transfection efficiency was 70–80%. The transfected HEL cells were treated with PMA and allowed to adhere to fibrinogen-coated coverslips, and cell spreading was measured after 30 min. The adherent cells were fixed and stained with Alexa 568 phalloidin, and the areas of cells were measured using ImageJ software; 300 cells were quantified per construct. Error bars, S.D. (p < 0.001). C, mouse RAW 264.7 cells were transiently transfected with plasmids encoding EGFP alone or EGFP-K2 constructs. The transfected cells were stained with 9EG7 monoclonal antibody to assess β1 integrin activation. Flow cytometry was used to measure 9EG7 binding. Error bars, S.E. of three independent experiments (p < 0.001). Intermediate flow cytometry data (dot plots and histograms) are shown in supplemental Fig. S2.

Article Snippet: The following primary antibodies were used: PAC1, which reacts with the activated but not the resting conformation of integrin αIIbβ3 ( 51 ) (BD Biosciences); 9EG7, which reacts selectively with the activated conformer of β1 integrins ( 41 ); mouse anti-EGFP (Takara Clontech, Mountain View, CA); mouse anti-GFP (clone B2) (Santa Cruz Biotechnology, Inc., Dallas, TX); mouse anti-β1 integrin (BD Transduction Laboratories); mouse anti-human CD41b (BD Biosciences); mouse anti-PSGL-1; clone KPL-1 (EMD Millipore, Temecula, CA); rabbit anti-integrin β3 integrin (Cell Signaling); mouse anti-GST (EMD Millipore); rabbit anti-PSGL-1(Santa Cruz Biotechnology); rabbit anti-kindlin 2 (Cell Signaling); rabbit anti-actin (Cell Signaling); and rabbit anti-ILK (Cell Signaling).

Techniques: Transfection, Activation Assay, Flow Cytometry, Expressing, Construct, Staining, Software, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: The extreme C-terminal region of kindlin-2 is critical to its regulation of integrin activation

doi: 10.1074/jbc.M117.776195

Figure Lengend Snippet: The C-terminal segment of kindlin-1 is necessary to maintain the co-activator function of K1 in talin-H mediated integrin activation in αIIbβ3-CHO cells. A, alignment of C-terminal sequences of human K1 and human K2. B, αIIbβ3-CHO cells were transiently transfected with plasmids encoding EGFP-fused K1, wild type or its mutants, and DsRed talin-H. After 24 h, activation of the integrin was quantified by flow cytometry as in Fig. 1. Error bars, S.D. Intermediate flow cytometry data (dot plots and histograms) are shown in supplemental Fig. S3.

Article Snippet: The following primary antibodies were used: PAC1, which reacts with the activated but not the resting conformation of integrin αIIbβ3 ( 51 ) (BD Biosciences); 9EG7, which reacts selectively with the activated conformer of β1 integrins ( 41 ); mouse anti-EGFP (Takara Clontech, Mountain View, CA); mouse anti-GFP (clone B2) (Santa Cruz Biotechnology, Inc., Dallas, TX); mouse anti-β1 integrin (BD Transduction Laboratories); mouse anti-human CD41b (BD Biosciences); mouse anti-PSGL-1; clone KPL-1 (EMD Millipore, Temecula, CA); rabbit anti-integrin β3 integrin (Cell Signaling); mouse anti-GST (EMD Millipore); rabbit anti-PSGL-1(Santa Cruz Biotechnology); rabbit anti-kindlin 2 (Cell Signaling); rabbit anti-actin (Cell Signaling); and rabbit anti-ILK (Cell Signaling).

Techniques: Activation Assay, Transfection, Flow Cytometry

Journal: The Journal of Biological Chemistry

Article Title: The extreme C-terminal region of kindlin-2 is critical to its regulation of integrin activation

doi: 10.1074/jbc.M117.776195

Figure Lengend Snippet: The C-terminal segment of kindlin-3 is dispensable for K3 functions. A, alignment of C-terminal sequences of human K2 and K3; B, αIIbβ3 integrin co-activation analysis using αIIbβ3-CHO cells transfected with EGFP-fused wild-type K3 or K3 chimera and talin-H; the activation of the integrin was determined after 24 h by flow cytometry with the activation-specific antibody PAC-1 (see “Experimental procedures”). Error bars, S.D. Intermediate flow cytometry data (dot plots and histograms) are shown in supplemental Fig. S4.

Article Snippet: The following primary antibodies were used: PAC1, which reacts with the activated but not the resting conformation of integrin αIIbβ3 ( 51 ) (BD Biosciences); 9EG7, which reacts selectively with the activated conformer of β1 integrins ( 41 ); mouse anti-EGFP (Takara Clontech, Mountain View, CA); mouse anti-GFP (clone B2) (Santa Cruz Biotechnology, Inc., Dallas, TX); mouse anti-β1 integrin (BD Transduction Laboratories); mouse anti-human CD41b (BD Biosciences); mouse anti-PSGL-1; clone KPL-1 (EMD Millipore, Temecula, CA); rabbit anti-integrin β3 integrin (Cell Signaling); mouse anti-GST (EMD Millipore); rabbit anti-PSGL-1(Santa Cruz Biotechnology); rabbit anti-kindlin 2 (Cell Signaling); rabbit anti-actin (Cell Signaling); and rabbit anti-ILK (Cell Signaling).

Techniques: Activation Assay, Transfection, Flow Cytometry

Journal: Scientific Reports

Article Title: Adaptation trajectories during adhesion and spreading affect future cell states

doi: 10.1038/s41598-017-12467-4

Figure Lengend Snippet: Differences and similarities in cell adhesion to the substrates ( a ) Heat map representation of gene expression levels (normalised scale) of cell adhesion receptors that were present on all samples and time points. ( b ) Integrin β1 adhesions are present on all gels after 24 h. Insets contain enlarged details of Integrin β1 adhesions. scale bar 50 µm.

Article Snippet: Following blocking with 10% BSA solution in PBS for 1 h, substrates were incubated with primary antibodies (vinculin (Abcam, ab18058), YAP/TAZ (Cell signalling, D24E4), β1 integrin (BD Biosciences, 550531), MyoD (BD Biosciences, 554130) and Alexa633-conjugated phalloidin (Sigma Aldrich) and subsequently with Alexa488 or -546-conjugated secondary antibodies (Life Technologies) and DAPI.

Techniques: Expressing

Journal: PLoS Pathogens

Article Title: Human-like PB2 627K Influenza Virus Polymerase Activity Is Regulated by Importin-α1 and -α7

doi: 10.1371/journal.ppat.1002488

Figure Lengend Snippet: (A and B) Polymerase activity of WSN-PB2 627K (A) or WSN-PB2 627E (B) containing vRNPs in importin-α siRNA silenced human 293T cells. Activity of vRNPs in negative siRNA silenced cells (Ctrl) was set 100%. As a background control, vRNPs were transfected omitting the PB2 subunit in negative siRNA silenced cells (Ctrl). Means of at least three independent experiments +/− standard deviations (SD) are shown (*p<0.05, **p<0.01, ***p<0.001, by students t -test). (C) Polymerase activity of WSN-PB2 627K or WSN-PB2 627E containing vRNPs in human 293T (left) and avian DF1 (right) cells. Mock transfected cells were used a control (Mock). (D) Confirmation of importin-α silencing in human 293T cells by Western blot analysis. GAPDH was used as a loading control.

Article Snippet: Primary antibodies used for Western blot analysis, cell fractionation and immunofluorescence assays include mouse anti-FLAG (Sigma), rabbit anti-importin-α1 (Abcam), goat anti-importin-α3 (Abcam), goat anti-importin-α4 (Abcam), rabbit anti-importin-α5/α7 (kindly provided by E. Hartmann, Institute of Biology, Lübeck, Germany), mouse anti-importin-β1 (BD Transduction Laboratories), rabbit anti-GAPDH and rabbit anti-LSD1 (Cell signaling), mouse anti-PB2 (kindly provided by J. Ortín, CSIC, Madrid, Spain), rabbit anti-PA (kindly provided by G.G.

Techniques: Activity Assay, Transfection, Western Blot

Journal: PLoS Pathogens

Article Title: Human-like PB2 627K Influenza Virus Polymerase Activity Is Regulated by Importin-α1 and -α7

doi: 10.1371/journal.ppat.1002488

Figure Lengend Snippet: (A) PB2 binding to endogenous importins. 293T cells were transfected with expression plasmids for PB2-627K-FLAG or PB2-627E-FLAG. Mock transfected cells served as a control (Mock). Cells were harvested 48 h after transfection and subjected to immunoprecipitation of FLAG-tagged PB2 proteins. The amount of co-immunoprecipitated endogenous importins (α1, α3, α4, α5, α7 and β1) was analyzed by Western blot. (B) Quantification of PB2 binding to endogenous importins in (A) using a Bioimager as described in Experimental Procedures. Precipitated amounts of importins were normalized against precipitated PB2 levels. Data shown are derived from at least three independent experiments and represent mean +/− SD. (C and D) PB2 binding to overexpressed importins. 293T cells were co-transfected with expression plasmids for FLAG-tagged importin-α proteins (α1, α3, α4, α5 or α7) and untagged PB2 627K (C) or PB2 627E (D). PB2-only transfected cells served as a control (Mock). Cells were lysed 48h after transfection and immunoprecipitated using the FLAG-tag. The amount of co-immunoprecipitated PB2 and importin-β1 was determined by Western blot analysis.

Article Snippet: Primary antibodies used for Western blot analysis, cell fractionation and immunofluorescence assays include mouse anti-FLAG (Sigma), rabbit anti-importin-α1 (Abcam), goat anti-importin-α3 (Abcam), goat anti-importin-α4 (Abcam), rabbit anti-importin-α5/α7 (kindly provided by E. Hartmann, Institute of Biology, Lübeck, Germany), mouse anti-importin-β1 (BD Transduction Laboratories), rabbit anti-GAPDH and rabbit anti-LSD1 (Cell signaling), mouse anti-PB2 (kindly provided by J. Ortín, CSIC, Madrid, Spain), rabbit anti-PA (kindly provided by G.G.

Techniques: Binding Assay, Transfection, Expressing, Immunoprecipitation, Western Blot, Derivative Assay, FLAG-tag

Journal: PLoS Pathogens

Article Title: Human-like PB2 627K Influenza Virus Polymerase Activity Is Regulated by Importin-α1 and -α7

doi: 10.1371/journal.ppat.1002488

Figure Lengend Snippet: (A) vRNP binding to endogenous importins. 293T cells were co-transfected with plasmids encoding PB2-627K-FLAG or PB2-627E-FLAG, PB1, PA, NP as well as the pPol-I-NP-Luc construct. Mock transfected cells were used a control (Mock). Cells were lysed 48 h after transfection and immunoprecipitation was performed using the FLAG-tag. The amount of co-immunoprecipitated PA, NP, importin-α1, -α3, -α4, -α5, -α7 and -β1 was analyzed by Western blotting. (B) Quantification of vRNP binding to endogenous importins in (A) using a Bioimager as described in Experimental Procedures. Amounts of Co-IP products were normalized against precipitated PB2 levels. Data shown represent the mean +/− SD of at least three independent experiments. (C) Trimeric polymerase binding to endogenous importins. Co-immunoprecipitation was performed as in (A), except NP expressing plasmid was omitted. (D) Quantification of trimeric polymerase binding to endogenous importins in (C) using a Bioimager as described in Experimental Procedures. Amounts of Co-IP products were normalized against precipitated PB2 levels. Data shown derived from four to six independent experiments and represent the mean +/− SD.

Article Snippet: Primary antibodies used for Western blot analysis, cell fractionation and immunofluorescence assays include mouse anti-FLAG (Sigma), rabbit anti-importin-α1 (Abcam), goat anti-importin-α3 (Abcam), goat anti-importin-α4 (Abcam), rabbit anti-importin-α5/α7 (kindly provided by E. Hartmann, Institute of Biology, Lübeck, Germany), mouse anti-importin-β1 (BD Transduction Laboratories), rabbit anti-GAPDH and rabbit anti-LSD1 (Cell signaling), mouse anti-PB2 (kindly provided by J. Ortín, CSIC, Madrid, Spain), rabbit anti-PA (kindly provided by G.G.

Techniques: Binding Assay, Transfection, Construct, Immunoprecipitation, FLAG-tag, Western Blot, Co-Immunoprecipitation Assay, Expressing, Plasmid Preparation, Derivative Assay

Journal: PLoS Pathogens

Article Title: Human-like PB2 627K Influenza Virus Polymerase Activity Is Regulated by Importin-α1 and -α7

doi: 10.1371/journal.ppat.1002488

Figure Lengend Snippet: (A) NP binding to endogenous importins. 293T cells were either Mock transfected or with plasmids encoding NP-FLAG and lysed 48 h after transfection. Immunoprecipitation was performed using the FLAG-tag. Amount of co-immunoprecipitated importin-α1, -α3, -α4, -α5, -α7 and -β1 was determined by Western blot analysis. (B) NP binding to overexpressed importins. 293T cells were co-transfected with plasmids encoding FLAG-tagged importins (α1, α3, α4, α5 or α7) and NP. NP-only transfected cells served as a control (Mock). Cells were lysed 48 h after transfection and immunoprecipitated using the FLAG-tag. The amount of co-immunoprecipitated NP and importin-β1 was determined by Western blot analysis.

Article Snippet: Primary antibodies used for Western blot analysis, cell fractionation and immunofluorescence assays include mouse anti-FLAG (Sigma), rabbit anti-importin-α1 (Abcam), goat anti-importin-α3 (Abcam), goat anti-importin-α4 (Abcam), rabbit anti-importin-α5/α7 (kindly provided by E. Hartmann, Institute of Biology, Lübeck, Germany), mouse anti-importin-β1 (BD Transduction Laboratories), rabbit anti-GAPDH and rabbit anti-LSD1 (Cell signaling), mouse anti-PB2 (kindly provided by J. Ortín, CSIC, Madrid, Spain), rabbit anti-PA (kindly provided by G.G.

Techniques: Binding Assay, Transfection, Immunoprecipitation, FLAG-tag, Western Blot

Journal: PLoS Pathogens

Article Title: Human-like PB2 627K Influenza Virus Polymerase Activity Is Regulated by Importin-α1 and -α7

doi: 10.1371/journal.ppat.1002488

Figure Lengend Snippet: (A–D) vRNP complexes containing either WSN-PB2-627K-FLAG or WSN-PB2-627E-FLAG were expressed in unsilenced controls (A), importin-α1 (B), -α3 (C), or -α7 (D) silenced 293T cells. Subcellular distribution of PB2 and NP was analyzed by specific staining against PB2-FLAG and NP protein. Mock transfected cells were used as negative controls.

Article Snippet: Primary antibodies used for Western blot analysis, cell fractionation and immunofluorescence assays include mouse anti-FLAG (Sigma), rabbit anti-importin-α1 (Abcam), goat anti-importin-α3 (Abcam), goat anti-importin-α4 (Abcam), rabbit anti-importin-α5/α7 (kindly provided by E. Hartmann, Institute of Biology, Lübeck, Germany), mouse anti-importin-β1 (BD Transduction Laboratories), rabbit anti-GAPDH and rabbit anti-LSD1 (Cell signaling), mouse anti-PB2 (kindly provided by J. Ortín, CSIC, Madrid, Spain), rabbit anti-PA (kindly provided by G.G.

Techniques: Staining, Transfection

Journal: PLoS Pathogens

Article Title: Human-like PB2 627K Influenza Virus Polymerase Activity Is Regulated by Importin-α1 and -α7

doi: 10.1371/journal.ppat.1002488

Figure Lengend Snippet: (A–D) Virus growth in importin silenced human cells. A549 cells were silenced using siRNA for importin-α1, -α3 or -α7 and infected at MOI of 0.001 with WSN-PB2 627K (A and B) or WSN-PB2 627E (C and D). Unsilenced (Mock) and control siRNA transfected cells (Ctrl) were used as a control. Virus titres were determined by plaque assay 72 and 96 hours post infection. As a further control negative siRNA silenced cells were infected (Ctrl). Data shown represent means of three to six independent experiments +/− standard deviations (SD) (*p<0.05, **p<0.01, ***p<0.001, by students t -test). (E) Confirmation of importin-α silencing in human A549 cells by Western blot analysis. GAPDH was used as a loading control.

Article Snippet: Primary antibodies used for Western blot analysis, cell fractionation and immunofluorescence assays include mouse anti-FLAG (Sigma), rabbit anti-importin-α1 (Abcam), goat anti-importin-α3 (Abcam), goat anti-importin-α4 (Abcam), rabbit anti-importin-α5/α7 (kindly provided by E. Hartmann, Institute of Biology, Lübeck, Germany), mouse anti-importin-β1 (BD Transduction Laboratories), rabbit anti-GAPDH and rabbit anti-LSD1 (Cell signaling), mouse anti-PB2 (kindly provided by J. Ortín, CSIC, Madrid, Spain), rabbit anti-PA (kindly provided by G.G.

Techniques: Infection, Transfection, Plaque Assay, Western Blot

Journal: PLoS Pathogens

Article Title: Human-like PB2 627K Influenza Virus Polymerase Activity Is Regulated by Importin-α1 and -α7

doi: 10.1371/journal.ppat.1002488

Figure Lengend Snippet: (A–F) Pathogenicity of human- and avian-like virus in wildtype and importin-α7 −/− mice. Wildtype ( n = 16) or importin-α7 −/− ( n = 16) mice were infected with 10 5 p.f.u. (∼20-fold MLD 50 ) of WSN-PB2 627K (A, C and E) or 5×10 6 p.f.u. (∼10-fold MLD 50 ) of WSN-PB2 627E (B, D and F). Survival (A and B) and weight loss (C and D) were monitored for 14 days. Mice receiving PBS were used as controls. Data shown representative average values per group. (E and F) Lungs were removed on days 3 ( n = 5) and 6 ( n = 5) post infection (p.i.). Virus titres were determined by plaque assay.

Article Snippet: Primary antibodies used for Western blot analysis, cell fractionation and immunofluorescence assays include mouse anti-FLAG (Sigma), rabbit anti-importin-α1 (Abcam), goat anti-importin-α3 (Abcam), goat anti-importin-α4 (Abcam), rabbit anti-importin-α5/α7 (kindly provided by E. Hartmann, Institute of Biology, Lübeck, Germany), mouse anti-importin-β1 (BD Transduction Laboratories), rabbit anti-GAPDH and rabbit anti-LSD1 (Cell signaling), mouse anti-PB2 (kindly provided by J. Ortín, CSIC, Madrid, Spain), rabbit anti-PA (kindly provided by G.G.

Techniques: Infection, Plaque Assay

Journal: bioRxiv

Article Title: Retrograde transport of mannose-6-phosphate receptor depends on several sorting machineries as analyzed by sulfatable nanobodies

doi: 10.1101/2019.12.21.885939

Figure Lengend Snippet: ( A ) Schematic representation of the functionalized nanobodies. The standard nanobody (VHH-std) consists of the GFP-specific VHH domain, T7 and HA epitope tags, a biotin acceptor peptide (BAP), and a hexahistidine (His6) purification tag. Other nanobodies in addition contain two tyrosine sulfation sequences (VHH-2xTS) or mCherry (VHH-mCherry). Scale bar in amino acids (aa). ( B ) Bacterially expressed and purified nanobodies (30 µ g) were analyzed by SDS-gel electrophoresis and Coomassie staining (left). Immunoblot analysis of nanobodies (10 ng) with antibodies against the HA, His6, T7, or mCherry epitopes, or with streptavidin-HRP (SA-HRP). Marker proteins with molecular weights in kDa are shown on the left. As previously reported ( ; ), mCherry-containing nanobodies are slightly susceptible to clipping between the VHH and mCherry domains. ( C ) HeLa cells were transfected with non-targeting siRNA or siRNAs targeting μ1A-adaptin, Vps26, TIP47, or Rab9. Three days after transfection, cells were subjected to immunoblot analysis with antibodies against the indicated proteins. ( D ) To determine the knockdown (kd) efficiency, the residual protein was quantified in percent of the value after control-kd (mean and standard deviation of three independent experiments). ( E ) HeLa cells stably expressing EGFP-CDMPR were depleted of μ1A-adaptin, Vps26, TIP47, or Rab9 as in (C). Cells were incubated for 1 h at 37°C with full medium containing 5 µ g/ml VHH-mCherry (∼0.1 µ M), fixed, stained for EEA1 and nuclei (DAPI, blue), and imaged by fluorescence microscopy. Bar: 10 µ m. ( F ) Quantitation of the percentage of cells displaying the CDMPR localization phenotypes “mainly TGN”, “mainly peripheral”, or “fully peripheral” as in and . For each condition, random frames with a total of 136–140 cells were scored from three independent experiments.

Article Snippet: For immunoblotting, mouse anti-α-adaptin (BD Biosciences #610501; 1:5000), mouse anti-γ-adaptin (BD Biosciences #610385; 1:5000), mouse anti-β1/2-adaptin (BD Biosciences #610381; 1:5000), mouse anti-γ-adaptin (made from 100/3 hybridoma; 1:5000), rabbit anti-σ1-adaptin (Bethyl Laboratories #A305-396A-M; 1:1000), mouse anti-γ-adaptin (made from 100/3 hybridoma; 1:5000), mouse anti-actin (EMD Millipore #MAB1501; 1:100000), mouse anti-CHC17 (made from TD.1 hybridoma; 1:200), rabbit anti-epsinR (Bethyl Laboratories #A301-926A; 1:1000), mouse anti-FLAG (Cell Signaling Technology #8146; 1:1000), mouse anti-GFP (Sigma-Aldrich #11814460001-Roche; 1:5000), rabbit anti-GGA1 (Bethyl Laboratories #A305-368A; 1:1000), mouse anti-GGA2 (BD Biosciences #612612; 1:2000), mouse anti-GGA3 (BD Biosciences #612310; 1:1000), mouse anti-HA (made from 12CA5 hybridoma; 1:10000), rabbit anti-His6 (Bethyl Laboratories #A190-114A; 1:10000), rabbit anti-mCherry (GeneTex #GTX128508; 1:10000), rabbit anti-T7 (Bethyl Laboratories #A190-117A; 1:10000), rabbit anti-Rab9 (Cell Signaling Technology #5118; 1:1000), mouse anti-SNX1 (BD Biosciences #611482; 1:500), rabbit anti-SNX2 (Bethyl Laboratories #A304-544A; 1:2000), rabbit anti-TIP47 (Proteintech 10694-1-AP; 1:1000), rabbit anti-Vps26 (Bethyl Laboratories #A304-801A; 1:1000), rabbit anti-Vps35 (Bethyl Laboratories #A304-727A; 1:1000), mouse anti-μ1A (Abnova H00008907-A01; 1:1000) antibodies were used.

Techniques: Purification, SDS-Gel, Electrophoresis, Staining, Western Blot, Marker, Transfection, Standard Deviation, Stable Transfection, Expressing, Incubation, Fluorescence, Microscopy, Quantitation Assay

Journal: bioRxiv

Article Title: Retrograde transport of mannose-6-phosphate receptor depends on several sorting machineries as analyzed by sulfatable nanobodies

doi: 10.1101/2019.12.21.885939

Figure Lengend Snippet: ( A ) HeLa cells stably expressing the sulfatable secretory protein α1-protease inhibitor (or anti-trypsin; SHMY-A1Pi; S, signal sequence of hemagglutinin; H, His6 epitope; M, myc epitope; Y, TS site) were transiently depleted of TIP47, Rab9, Vps26, μ1A-adaptin, epsinR, and GGAs (GGA1–3) for 72 h. Cells were labeled with [ 35 S]sulfate labeling for 75 min, lysed, HMY-A1Pi was isolated by Ni/NTA beads, and subjected to SDS-gel electrophoresis followed by autoradiography ([ 35 S]). In parallel, aliquots of the cell lysates were immunoblotted for actin as a control for the amount of cells used. ( B ) Quantitation of total tyrosine sulfation as judged by sulfation of the secretory reporter HMY-A1Pi as in panel (A) is shown in percent of the value present in control-kd (mean and standard deviation of three independent experiments).

Article Snippet: For immunoblotting, mouse anti-α-adaptin (BD Biosciences #610501; 1:5000), mouse anti-γ-adaptin (BD Biosciences #610385; 1:5000), mouse anti-β1/2-adaptin (BD Biosciences #610381; 1:5000), mouse anti-γ-adaptin (made from 100/3 hybridoma; 1:5000), rabbit anti-σ1-adaptin (Bethyl Laboratories #A305-396A-M; 1:1000), mouse anti-γ-adaptin (made from 100/3 hybridoma; 1:5000), mouse anti-actin (EMD Millipore #MAB1501; 1:100000), mouse anti-CHC17 (made from TD.1 hybridoma; 1:200), rabbit anti-epsinR (Bethyl Laboratories #A301-926A; 1:1000), mouse anti-FLAG (Cell Signaling Technology #8146; 1:1000), mouse anti-GFP (Sigma-Aldrich #11814460001-Roche; 1:5000), rabbit anti-GGA1 (Bethyl Laboratories #A305-368A; 1:1000), mouse anti-GGA2 (BD Biosciences #612612; 1:2000), mouse anti-GGA3 (BD Biosciences #612310; 1:1000), mouse anti-HA (made from 12CA5 hybridoma; 1:10000), rabbit anti-His6 (Bethyl Laboratories #A190-114A; 1:10000), rabbit anti-mCherry (GeneTex #GTX128508; 1:10000), rabbit anti-T7 (Bethyl Laboratories #A190-117A; 1:10000), rabbit anti-Rab9 (Cell Signaling Technology #5118; 1:1000), mouse anti-SNX1 (BD Biosciences #611482; 1:500), rabbit anti-SNX2 (Bethyl Laboratories #A304-544A; 1:2000), rabbit anti-TIP47 (Proteintech 10694-1-AP; 1:1000), rabbit anti-Vps26 (Bethyl Laboratories #A304-801A; 1:1000), rabbit anti-Vps35 (Bethyl Laboratories #A304-727A; 1:1000), mouse anti-μ1A (Abnova H00008907-A01; 1:1000) antibodies were used.

Techniques: Stable Transfection, Expressing, Protease Inhibitor, Sequencing, Labeling, Isolation, SDS-Gel, Electrophoresis, Autoradiography, Quantitation Assay, Standard Deviation

Journal: bioRxiv

Article Title: Retrograde transport of mannose-6-phosphate receptor depends on several sorting machineries as analyzed by sulfatable nanobodies

doi: 10.1101/2019.12.21.885939

Figure Lengend Snippet: ( A and B ) Cells stably expressing EGFP-CDMPR were transfected with non-targeting siRNA (control-kd) or with siRNA silencing expression of μ1A as described in . The cells were labeled with [ 35 S]sulfate for up to 75 min in the presence of 2 μg/ml VHH-2xTS and the nanobodies were isolated, analyzed, and quantified as in (mean and standard deviation of three independent experiments; two-sided Student’s t -test: *, p < 0.05; **, p < 0.01). Control-kd is shown as black squares and μ1A-kd as gray circles; uptake as open symbols, sulfation as filled symbols. ( C ) Immunoblot analysis of parental HeLa cells (HeLa wt) and of a pool of γ-adaptin-knockout cells (AP-1γ-ko) generated with CRISPR/Cas9. Equal amounts of cell lysates were probed with antibodies against specific AP-1 subunits (γ, μ1A, σ1), β-adaptins of AP-1 and AP-2 (β1/2), AP-2α, clathrin heavy-chain (CHC17), and actin. ( D ) Parental HeLa cells and AP-1γ knockout cells were mixed and stained with antibodies targeting AP-1γ or GM130. γ-Adaptin staining was completely absent in knockout cells, while Golgi morphology remained intact. ( E ) AP-1γ knockout cells were transiently transfected with γ-FKBP (a fusion protein of γ-adaptin with FK506 binding protein; see Materials and Methods) and incubated with anti-CIMPR antibody for 1 h at 37°C. Cells were fixed and prepared for immunofluorescence microscopy by staining the recombinant γ-subunit and the internalized antibody. Non-transfected cells mostly displayed peripheral accumulation of anti-CIMPR antibody, while expression of γ-FKBP largely rescued perinuclear anti-CIMPR antibody localization. Nuclei were stained with DAPI (blue). Bar: 10 µ m. ( F and G ) Parental HeLa and AP-1γ knockout cells were transiently transfected with EGFP-CDMPR, followed by [ 35 S]sulfate labeling for 75 min in the presence of 2 µ g/ml VHH-2xTS. The nanobodies were isolated, analyzed, and quantified as in (mean and standard deviation of three independent experiments).

Article Snippet: For immunoblotting, mouse anti-α-adaptin (BD Biosciences #610501; 1:5000), mouse anti-γ-adaptin (BD Biosciences #610385; 1:5000), mouse anti-β1/2-adaptin (BD Biosciences #610381; 1:5000), mouse anti-γ-adaptin (made from 100/3 hybridoma; 1:5000), rabbit anti-σ1-adaptin (Bethyl Laboratories #A305-396A-M; 1:1000), mouse anti-γ-adaptin (made from 100/3 hybridoma; 1:5000), mouse anti-actin (EMD Millipore #MAB1501; 1:100000), mouse anti-CHC17 (made from TD.1 hybridoma; 1:200), rabbit anti-epsinR (Bethyl Laboratories #A301-926A; 1:1000), mouse anti-FLAG (Cell Signaling Technology #8146; 1:1000), mouse anti-GFP (Sigma-Aldrich #11814460001-Roche; 1:5000), rabbit anti-GGA1 (Bethyl Laboratories #A305-368A; 1:1000), mouse anti-GGA2 (BD Biosciences #612612; 1:2000), mouse anti-GGA3 (BD Biosciences #612310; 1:1000), mouse anti-HA (made from 12CA5 hybridoma; 1:10000), rabbit anti-His6 (Bethyl Laboratories #A190-114A; 1:10000), rabbit anti-mCherry (GeneTex #GTX128508; 1:10000), rabbit anti-T7 (Bethyl Laboratories #A190-117A; 1:10000), rabbit anti-Rab9 (Cell Signaling Technology #5118; 1:1000), mouse anti-SNX1 (BD Biosciences #611482; 1:500), rabbit anti-SNX2 (Bethyl Laboratories #A304-544A; 1:2000), rabbit anti-TIP47 (Proteintech 10694-1-AP; 1:1000), rabbit anti-Vps26 (Bethyl Laboratories #A304-801A; 1:1000), rabbit anti-Vps35 (Bethyl Laboratories #A304-727A; 1:1000), mouse anti-μ1A (Abnova H00008907-A01; 1:1000) antibodies were used.

Techniques: Stable Transfection, Expressing, Transfection, Labeling, Isolation, Standard Deviation, Western Blot, Knock-Out, Generated, CRISPR, Staining, Binding Assay, Incubation, Immunofluorescence, Microscopy, Recombinant

Journal: bioRxiv

Article Title: Retrograde transport of mannose-6-phosphate receptor depends on several sorting machineries as analyzed by sulfatable nanobodies

doi: 10.1101/2019.12.21.885939

Figure Lengend Snippet: ( A ) Lysates of normal HeLa cells and HeLa-AP1 knocksideways (HeLa-AP1ks) cells stably expressing γ-FKBP, Mitotrap and EGFP-CDMPR with or without siRNA-mediated knockdown of the endogenous γ-adaptin were subjected to immunoblot analysis for both forms of γ-adaptin, for Mitotrap (anti-FLAG), EGFP-CDMPR, the α-adaptin subunit of AP-2, and clathrin heavy-chain (CHC17). Knockdown efficiencies for endogenous γ-adaptin were typically >85%. ( B ) HeLa-AP1ks cells stably expressing EGFP-CDMPR after silencing endogenous γ-adaptin were treated with or without 500 nM rapamycin for 1 h and processed for fluorescence microscopy to detect EGFP-CDMPR, recombinant γ-FKBP (using an antibody targeting an epitope present in the neuronal splice variant of AP-2α), and Mitotrap (anti-FLAG). Bar: 10 µ m. ( C ) HeLa-AP1ks cells stably expressing EGFP-CDMPR were siRNA-silenced for endogenous γ-adaptin, followed by starvation for sulfate in the presence of VHH-2xTS. The cells were then labeled with [ 35 S]sulfate for up to 75 min, without or with addition of 500 nM rapamycin after 15 min (arrow) to inactivate AP-1 (+rapa). The nanobodies were isolated by Ni/NTA beads and subjected to SDS-gel electrophoresis followed by immunoblot analysis (anti-His6) and autoradiography ([ 35 S]). In parallel, aliquots of the cell lysates were immunoblotted for actin as a control for the amount of cells used. ( D ) Three independent experiments as shown in panels C were quantified and presented as the percentage of the value in the absence of rapamycin after 75 min (mean and standard deviation of three independent experiments; two-sided Student’s t -test: *, p < 0.05; **, p < 0.01). Without rapamycin is shown as black squares, with rapamycin as gray circles; uptake as open symbols, sulfation as filled symbols.

Article Snippet: For immunoblotting, mouse anti-α-adaptin (BD Biosciences #610501; 1:5000), mouse anti-γ-adaptin (BD Biosciences #610385; 1:5000), mouse anti-β1/2-adaptin (BD Biosciences #610381; 1:5000), mouse anti-γ-adaptin (made from 100/3 hybridoma; 1:5000), rabbit anti-σ1-adaptin (Bethyl Laboratories #A305-396A-M; 1:1000), mouse anti-γ-adaptin (made from 100/3 hybridoma; 1:5000), mouse anti-actin (EMD Millipore #MAB1501; 1:100000), mouse anti-CHC17 (made from TD.1 hybridoma; 1:200), rabbit anti-epsinR (Bethyl Laboratories #A301-926A; 1:1000), mouse anti-FLAG (Cell Signaling Technology #8146; 1:1000), mouse anti-GFP (Sigma-Aldrich #11814460001-Roche; 1:5000), rabbit anti-GGA1 (Bethyl Laboratories #A305-368A; 1:1000), mouse anti-GGA2 (BD Biosciences #612612; 1:2000), mouse anti-GGA3 (BD Biosciences #612310; 1:1000), mouse anti-HA (made from 12CA5 hybridoma; 1:10000), rabbit anti-His6 (Bethyl Laboratories #A190-114A; 1:10000), rabbit anti-mCherry (GeneTex #GTX128508; 1:10000), rabbit anti-T7 (Bethyl Laboratories #A190-117A; 1:10000), rabbit anti-Rab9 (Cell Signaling Technology #5118; 1:1000), mouse anti-SNX1 (BD Biosciences #611482; 1:500), rabbit anti-SNX2 (Bethyl Laboratories #A304-544A; 1:2000), rabbit anti-TIP47 (Proteintech 10694-1-AP; 1:1000), rabbit anti-Vps26 (Bethyl Laboratories #A304-801A; 1:1000), rabbit anti-Vps35 (Bethyl Laboratories #A304-727A; 1:1000), mouse anti-μ1A (Abnova H00008907-A01; 1:1000) antibodies were used.

Techniques: Stable Transfection, Expressing, Western Blot, Fluorescence, Microscopy, Recombinant, Variant Assay, Labeling, Isolation, SDS-Gel, Electrophoresis, Autoradiography, Standard Deviation

Journal:

Article Title: Pathogenic Mechanism of Mouse Brain Damage Caused by Oral Infection with Shiga Toxin-Producing Escherichia coli O157:H7

doi:

Figure Lengend Snippet: Demonstration of functional receptors for Stx2 in the neutral glycolipid fraction of mouse brain. Standard mixtures of neutral glycolipids (lane A) and neutral glycolipids of the mouse brain (lane B) were separated by TLC and visualized with orcinol reagent. Glycolipids cross-reacting with the epitope of Gb3 were identified by immunostaining on TLC-blotted PVDF membranes (lane C from the blot in lane A; lane D from the blot in lane B) with an anti-Pk MAb, followed by treatment with peroxidase-conjugated goat anti-mouse IgM F(ab′)2 and DAB-H2O2. The TLC-blotted membranes used for lane D was incubated with 1 μg of Stx2 per ml; and glycolipids to which Stx2 bound were visualized by immunostaining with an anti-Stx2 MAb, followed by treatment with biotinylated anti-mouse IgG F(ab′)2, avidin-peroxidase complex, and DAB-H2O2 (lane E).

Article Snippet: To examine Gb3, the blot membrane was washed with TNMC after blocking and overlaid with an anti-P k monoclonal antibody (MAb) (specificity, Galα 1-4Gal β1-4 Glcβ1-1Cer moiety of Gb3; isotype, murine immunoglobulin M [IgM]) (Accurate Chemical & Scientific Corporation) ( 1 ) diluted 1:8 in 10% horse serum in TNMC.

Techniques: Functional Assay, Immunostaining, Incubation, Avidin-Biotin Assay

Journal: PLoS ONE

Article Title: Imaging the Impact of Chemically Inducible Proteins on Cellular Dynamics In Vivo

doi: 10.1371/journal.pone.0030177

Figure Lengend Snippet: A) Expression vectors encoding tunable zsGreen (pzsGreen-DD), fluorescent E-cadherin (pE-cadh-zsG) and tunable fluorescent E-cadherin (pE-cadh-zsG-DD). Components include CMV promoter (pCMV), zsGreen fluorescent protein (zsGreen), the Shield-1 binding degradation domain (FKBP-DD), and E-cadherin. B) Schematic of MDA-MB-231-luc-D3H2LN (231LN) cells used to express tunable proteins and the predicted behavior of cells in the presence or absence of Shield-1. 231LN tumor cells were stably transfected with tdTomato and zsGreen alone or as a fusion with E-cadherin. C) Intravital imaging platform (right) with avian embryo imaging chamber (left) to maintain proper temperature (37°C) and humidity (>90%) used to perform in vivo three dimensional time-lapse imaging of micrometastases in the chorioallantoic membrane of the avian embryo.

Article Snippet: The monoclonal antibody for E-cadherin was from BD Pharmingen (Burlington, ON), the monoclonal antibody for vimentin was from Dako (clone 3B4, Burlington, ON), the monoclonal antibody to detect 20S proteasome was Subunit β1, clone MCP421 from Biomol (Burlington, ON), and the Goat anti-mouse Alexa647 secondary antibody and Hoechst 33345 were from Invitrogen (San Diego, CA).

Techniques: Expressing, Binding Assay, Stable Transfection, Transfection, Imaging, In Vivo, Membrane

Journal: PLoS ONE

Article Title: Imaging the Impact of Chemically Inducible Proteins on Cellular Dynamics In Vivo

doi: 10.1371/journal.pone.0030177

Figure Lengend Snippet: 231LN cells containing both tdTomato and zsGreen-DD were grown on glass coverslips. Panels represent fluorescence time-lapse imaging of 231LN cells treated with vehicle (A) and 1.0 µM of Shield (B). C) Quantification of zsGreen signal within the cells in the presence and absence of Shield-1 over time (*denotes p<0.01 compared to Vehicle treatment kinetic, N>10 cells per field of view, 10 fields of view analyzed per group). Treatment with 0.5, 1.0 and 2.0 µM Shield-1 revealed similar first order kinetics, while treatment with 0.2 µM Shield-1 revealed a similarly steep but brief increase (induction) in signal accumulation followed by a less steep kinetic at 4 hours post-treatment (depletion kinetic). D) Fluorescence immunohistochemistry demonstrates co-localization of proteasome (α1-20S antibody in red) with zsGreen-DD signal in 231LN cells in the absence of Shield-1. All scale bars are 25 µm.

Article Snippet: The monoclonal antibody for E-cadherin was from BD Pharmingen (Burlington, ON), the monoclonal antibody for vimentin was from Dako (clone 3B4, Burlington, ON), the monoclonal antibody to detect 20S proteasome was Subunit β1, clone MCP421 from Biomol (Burlington, ON), and the Goat anti-mouse Alexa647 secondary antibody and Hoechst 33345 were from Invitrogen (San Diego, CA).

Techniques: Fluorescence, Imaging, Immunohistochemistry

Journal: PLoS ONE

Article Title: Imaging the Impact of Chemically Inducible Proteins on Cellular Dynamics In Vivo

doi: 10.1371/journal.pone.0030177

Figure Lengend Snippet: A) Representative images of 231LN cells expressing fluorescent E-cadherin chimeras. Cell nucleus as stained by Hoechst (blue), E-cadherin-zsGreen (green), and tdTomato to highlight the cytoplasm (red) reveal the changes in cell morphology when E-cadherin is over-expressed (row 2) or induced with Shield-1 for 12 hours (row 4) compared to control (row 1) or un-induced cells (row 3). Arrows (yellow) highlight junctions formed by Shield-1-stabilized E-cadh-zG-DD. Scale bars are 20 µm. Insets show magnified view (250%) of cellular junctions. B) Examples of circularity measurements of representative 231LN cells (left) and 231LN cells expressing E-cadh-zsG-DD treated with 1.0 µM Shield-1 (right). C) Circularity measurements to assess a mesenchymal vs. epithelial morphology in cells described above. N = 70 per group, * denotes p<0.01 between groups, 2-way ANOVA.

Article Snippet: The monoclonal antibody for E-cadherin was from BD Pharmingen (Burlington, ON), the monoclonal antibody for vimentin was from Dako (clone 3B4, Burlington, ON), the monoclonal antibody to detect 20S proteasome was Subunit β1, clone MCP421 from Biomol (Burlington, ON), and the Goat anti-mouse Alexa647 secondary antibody and Hoechst 33345 were from Invitrogen (San Diego, CA).

Techniques: Expressing, Staining, Control

Journal: PLoS ONE

Article Title: Imaging the Impact of Chemically Inducible Proteins on Cellular Dynamics In Vivo

doi: 10.1371/journal.pone.0030177

Figure Lengend Snippet: A) 231LN cells expressing E-cadh-zsG-DD (green) treated with 1.0 µM Shield-1 for 24 hours and immunostained with anti-E-cadherin mAb (red) and Hoechst nuclear stain (blue). Scale bars are 25 µm. B) Western immunoblot analysis of E-cadherin expression in 231LN cells expressing E-cadh-zsG-DD and treated with 1.0 µM Shield-1 using the same mAb as in A). Graph (right) represents analyses performed on three independent induction experiments. Cell lysates of 231LN cells expressing E-cadherin-zsG are shown in the first lane. Lysates of cells expressing E-cadherin-zsG-DD were collected at 0, 4, 8, 12, 16, and 24 hrs after Shield-1 treatment (1.0 µM final), revealing accumulation of Shield-1 stabilized E-cadherin-zsG-DD within cells (∼135 kDa). Far right lane is a positive control of 21PT cells which endogenously express high levels of E-cadherin (∼110 kDa). C) Western immunoblot analysis of markers for epithelial-mesenchymal transition (EMT). Blot (left panels) reveals a decrease in vimentin protein levels when E-cadh-zsG-DD is induced by 1.0 µM Shield-1 treatment. Graph (right) represents analyses performed on three independent induction experiments.

Article Snippet: The monoclonal antibody for E-cadherin was from BD Pharmingen (Burlington, ON), the monoclonal antibody for vimentin was from Dako (clone 3B4, Burlington, ON), the monoclonal antibody to detect 20S proteasome was Subunit β1, clone MCP421 from Biomol (Burlington, ON), and the Goat anti-mouse Alexa647 secondary antibody and Hoechst 33345 were from Invitrogen (San Diego, CA).

Techniques: Expressing, Staining, Western Blot, Positive Control

Journal: PLoS ONE

Article Title: Imaging the Impact of Chemically Inducible Proteins on Cellular Dynamics In Vivo

doi: 10.1371/journal.pone.0030177

Figure Lengend Snippet: Fluorescence in vitro time-lapse imaging of 231LN cells containing inducible E-cadherin treated with vehicle (A) or 0.2 µM Shield-1 which will produce an induction and depletion effect over 24 hours (B). Data Scale bar is 25 µm. C) Measure of “actively engaged" E-cadherin in 231LN cells in the presence of varying levels of Shield-1 (0.2, 0.5, and 1.0 µM Shield-1), expressed as the cumulative length of all zsG-positive adherens junctions over time (µm/hrs) in representative time-lapse experiments. The black kinetic represents the total cumulative length of E-cadherin-based junctions within a field of view at that timepoint while the red kinetic represents the accumulation of zsGreen-DD exposed to similar Shield-1 treatment. The “induction” and “depletion” phases of chemical induction are annotated in each graph. D) Conditioned media collected from cells expressing pzsGreen-DD which were treated with 0.2 µM Shield for 0, 6 and 12 hours were used to induce E-cadherin-zsGreen-DD expression in 231LN cells expressing E-cad-zsG-DD. There is induction with the 0 and 6 hours conditioned media, but minimal effect with the 12 hour conditioned media. Conversely, conditioned media from cells treated with 5.0 µM Shield-1 induced E-cadherin-zsG-DD expression regardless of the time of conditioned media collection. Graph ( , right) represents data from three independent western immunoblot experiments.

Article Snippet: The monoclonal antibody for E-cadherin was from BD Pharmingen (Burlington, ON), the monoclonal antibody for vimentin was from Dako (clone 3B4, Burlington, ON), the monoclonal antibody to detect 20S proteasome was Subunit β1, clone MCP421 from Biomol (Burlington, ON), and the Goat anti-mouse Alexa647 secondary antibody and Hoechst 33345 were from Invitrogen (San Diego, CA).

Techniques: Fluorescence, In Vitro, Imaging, Expressing, Western Blot

Journal: PLoS ONE

Article Title: Imaging the Impact of Chemically Inducible Proteins on Cellular Dynamics In Vivo

doi: 10.1371/journal.pone.0030177

Figure Lengend Snippet: 231LN cells expressing tdTomato (red) and inducible zsGreen-DD (green) were injected intravenously in the avian embryo and allowed to extravasate and proliferate into micrometastases. Representative time-lapse images (maximum intensity projections) are shown after intravenous administration of Vehicle (A), 0.2 µM Shield-1 (B), and 0.5 µM Shield-1 (C). D) Quantification of in vivo zsGreen fluorescence in tdTomato-positive cells over time. Data for Vehicle (black kinetic), 0.2 µM Shield-1 (red kinetic), 0.5 µM Shield-1 (green kinetic), and 1.0 µM Shield-1 (blue kinetic) are represented as averages of at least three movies analyzed in each group. Error bars are SE and scale bar represents 25 µm.

Article Snippet: The monoclonal antibody for E-cadherin was from BD Pharmingen (Burlington, ON), the monoclonal antibody for vimentin was from Dako (clone 3B4, Burlington, ON), the monoclonal antibody to detect 20S proteasome was Subunit β1, clone MCP421 from Biomol (Burlington, ON), and the Goat anti-mouse Alexa647 secondary antibody and Hoechst 33345 were from Invitrogen (San Diego, CA).

Techniques: Expressing, Injection, In Vivo, Fluorescence

Journal: PLoS ONE

Article Title: Imaging the Impact of Chemically Inducible Proteins on Cellular Dynamics In Vivo

doi: 10.1371/journal.pone.0030177

Figure Lengend Snippet: 231LN cells expressing tdTomato (red) and tunable E-cadherin-zsGreen-DD (green) were injected intravenously in the avian embryo and allowed to extravasate and proliferate into micrometastases. Representative maximum intensity projections are shown. A) In vivo treatment with 1.0 µM Shield-1 demonstrates transition from a mesenchymal morphology to an epithelial morphology and continued maintenance of the epithelial morphology over an extended period of time (>40 hrs). Formation of E-cadherin junctions is apparent at t = 0.5 hrs, increasing through 24 hrs. B) Representative micrometastatic colony expressing tunable E-cadherin-zsG-DD and treated with 0.2 µM Shield-1. No induction effect is observed with 0.2 µM Shield-1 in vivo . C) Single Z-plane slices of a representative micrometastastic colony expressing tunable E-cadherin-zsG-DD and treated with 0.5 µM Shield-1. These panels represent the stabilization effect induced by 0.5 µM Shield-1 over the 12 hour time course; E-cadh-zsG-DD is stabilized and junctions appear between 231LN cells. E-cadh-zsG-DD junctions between cells of the micrometastatic colony are highlighted by arrows. D) Panels represent the depletion effect in the same colony with depleted levels of 0.5 µM Shield-1; E-cadh-zsG-DD junctions gradually disperse over time and 231LN cells eventually revert to a mesenchymal morphology. All scale bars are 25 µm. E) Quantitation of E-cadh-zsG-DD signal in 231LN-tdTomato cells in a 4-dimension image set over the entire 28 hour time course.

Article Snippet: The monoclonal antibody for E-cadherin was from BD Pharmingen (Burlington, ON), the monoclonal antibody for vimentin was from Dako (clone 3B4, Burlington, ON), the monoclonal antibody to detect 20S proteasome was Subunit β1, clone MCP421 from Biomol (Burlington, ON), and the Goat anti-mouse Alexa647 secondary antibody and Hoechst 33345 were from Invitrogen (San Diego, CA).

Techniques: Expressing, Injection, In Vivo, Quantitation Assay

Journal: Oncotarget

Article Title: Structural composition of components of geoherb Moutan Cortex contributes to anti-diabetic nephropathy activity

doi: 10.18632/oncotarget.23771

Figure Lengend Snippet: Figure 1: Comparison on the down-regulation of Moutan Cortex from different regions on protein expression levels of ICAM-1,TGF-β1 and FN. HBZY-1 mesangial cells were treated with 200 μg/mL AGEs in the presence or absence of Moutan Cortex (MC) extract of 200 μg/mL. Aminoguanidine of 10 μM was used as the positive control while BSA (200 μg/mL) as blank control. (A) Western blotting was performed to compare the protein expression levels. (B–D) The grayscale scan results of ICAM-1, TGF-β1 and FN. a, Control; b, 200 μg/mL AGEs; c, Positive control aminoguanidine group; “d-m” represent MC from Anhui, Guizhou, Zhejiang, Henan, Hunan, Hebei, Sichuan, Chongqing, Shandong, Gansu. Data are expressed as means ± SD, n = 3. ###P < 0.001 vs. BSA group; *P < 0.05, **P < 0.01 and ***P < 0.001 vs. AGEs group; $P < 0.05, $$P < 0.01 and $$$P < 0.001 vs. MC from Anhui group.

Article Snippet: Rabbit anti-mouse FN, TGF-β1 and ICAM-1 monoclonal antibodies were offered by Boster Biological Engineering Co., Ltd. (Wuhan, China).

Techniques: Comparison, Expressing, Positive Control, Control, Western Blot

Journal: Oncotarget

Article Title: Structural composition of components of geoherb Moutan Cortex contributes to anti-diabetic nephropathy activity

doi: 10.18632/oncotarget.23771

Figure Lengend Snippet: Figure 2: Effect of Moutan Cortex from different regions on ICAM-1 and TGF-β1 protein expression levels in kidney of DN rats. After being treated with STZ and/or MC extract of 5g/kg or , immunohistochemistry was conducted to evaluate the expression levels of ICAM-1 (A) and TGF-β1 (B) of renal tissues. “a” represents normal control; “b” represents model group (DN rats); “c” represents Positive control 0.1 g/kg AG; “d-m” represent Gansu, Chongqing, Shangdong, Sichuan, Zhejiang, Anhui, Hunan, Guizhou, Hebei, Henan.

Article Snippet: Rabbit anti-mouse FN, TGF-β1 and ICAM-1 monoclonal antibodies were offered by Boster Biological Engineering Co., Ltd. (Wuhan, China).

Techniques: Expressing, Immunohistochemistry, Control, Positive Control