Journal: PLoS ONE

Article Title: Leucine zipper transcription factor-like 1 binds adaptor protein complex-1 and 2 and participates in trafficking of transferrin receptor 1

doi: 10.1371/journal.pone.0226298

Figure Lengend Snippet: (A) Schematic representation of potential protein-binding domains of LZTFL1. (B) Purified LZTFL1 fused with maltose-binding protein (MBP, left ) as well as individual subunits of AP-1 complex (β1, γ, μ1, and σ1) fused with glutathione S-transferase (GST, right ) were visualized by Coomassie Brilliant Blue (CBB) staining. Full-length GST-β1 and GST-γ are indicated with red asterisks. (C) Purified MBP or MBP-LZTFL1 was incubated with each of the purified GST-fused subunits of the AP-1 complex (β1, γ, μ1, and σ1), and the LZTFL1-bound subunit was purified by amylose resin and analyzed by western blotting using anti-MBP and anti-GST antibodies. The GST-β1 band is indicated with a red asterisk. (D, top) Purified wild-type LZTFL1 and LZTFL1 mutants fused with MBP and (D, bottom) β1 and β2 subunits of AP-1 and AP-2 respectively fused with GST were visualized by CBB staining. Purified MBP, MBP-LZTFL1, or mutants were incubated with (E) GST-β1 (AP-1) or (F) GST-β2 (AP-2) and pulled-down proteins were analyzed by western blotting using anti-MBP and anti-GST antibodies. (G) Brain tissue lysate from wild-type and Lztfl1 -knockout mice was analyzed by western blotting using anti-LZTFL1 antibody, anti-γ subunit of AP-1 antibody, anti-α subunit of AP-2 antibody, and REVERT staining for total protein (left) . Lysate was immunoprecipitated with anti-LZTFL1-conjugated beads. The LZTFL1-interacting proteins were eluted by NuPAGE LDS buffer and analyzed by western blotting using anti-LZTFL1, anti-γ subunit of AP-1, and anti-α subunit of AP-2 antibodies (right) . (H, left) Lysate from HEK293FT cells transiently expressing FLAG or LZTFL1-FLAG was analyzed by western blotting using anti-FLAG, anti-β1 subunit and anti-γ subunit of AP-1, and anti-β-actin antibodies or (I, left) anti-FLAG, anti-α subunit of AP-2, and anti-actin antibodies. (H, right) Cell lysate was immunoprecipitated with anti-FLAG M2 magnetic beads. The LZTFL1-interacting proteins were eluted by 3X FLAG peptide and analyzed by western blotting using anti-FLAG, anti-β1 subunit of AP-1, and anti-γ subunit of AP-1 antibodies or (I, right) anti-FLAG and anti-α subunit of AP-2 antibodies.

Article Snippet: Plasmids expressing human LZTFL1 (NM_020347) and TfR1 (NM_003234) with Myc-FLAG tag in pCMV6-Entry vector were purchased from OriGene Technologies (Rockville, MD).

Techniques: Protein Binding, Purification, Binding Assay, Staining, Incubation, Western Blot, Knock-Out, Immunoprecipitation, Expressing, Magnetic Beads

Journal: PLoS ONE

Article Title: Leucine zipper transcription factor-like 1 binds adaptor protein complex-1 and 2 and participates in trafficking of transferrin receptor 1

doi: 10.1371/journal.pone.0226298

Figure Lengend Snippet: (A) Schematic representation of wild-type and LZTFL1 mutants. (B) Cell lysate from HEK293FT cells expressing FLAG-tagged wild-type LZTFL1 or the indicated mutants was analyzed by western blotting using anti-FLAG, anti-γ subunit of AP-1, anti-β1 subunit of AP-1, and anti-β-actin antibodies. Cell lysate was immunoprecipitated with anti-FLAG M2 magnetic beads. The interacting proteins were eluted by 3X FLAG peptide and analyzed by western blotting using anti-FLAG, anti-γ subunit of AP-1, and anti-β1 subunit of AP-1 antibodies. (C) Lysate from HEK293FT cells transiently expressing GFP-FLAG, LZTFL1-FLAG, or the indicated mutants was analyzed by western blotting using anti-FLAG, anti-α subunit of AP-2, and anti-tubulin antibodies. Lysate was immunoprecipitated and analyzed by western blotting using anti-FLAG and anti-α subunit of AP-2 antibodies.

Article Snippet: Plasmids expressing human LZTFL1 (NM_020347) and TfR1 (NM_003234) with Myc-FLAG tag in pCMV6-Entry vector were purchased from OriGene Technologies (Rockville, MD).

Techniques: Expressing, Western Blot, Immunoprecipitation, Magnetic Beads

Journal: PLoS ONE

Article Title: Leucine zipper transcription factor-like 1 binds adaptor protein complex-1 and 2 and participates in trafficking of transferrin receptor 1

doi: 10.1371/journal.pone.0226298

Figure Lengend Snippet: (A) HeLa cells were incubated with or without 7.5 μg/ml BFA for 2 minutes, washed with complete DMEM to remove BFA, and incubated with complete DMEM for 0, 15, 30, and 60 minutes. Cells were fixed and stained with antibodies to LZTFL1 (green), γ subunit of AP-1 (red) and p230 (TGN marker, magenta). Scale bar = 10 μm. (B) Quantitative analysis of the colocalization of LZTFL1 and AP-1 at the TGN (mean + SD). Student’s t-test, n>30 cells, ***p<0.001.

Article Snippet: Plasmids expressing human LZTFL1 (NM_020347) and TfR1 (NM_003234) with Myc-FLAG tag in pCMV6-Entry vector were purchased from OriGene Technologies (Rockville, MD).

Techniques: Incubation, Staining, Marker

Journal: PLoS ONE

Article Title: Leucine zipper transcription factor-like 1 binds adaptor protein complex-1 and 2 and participates in trafficking of transferrin receptor 1

doi: 10.1371/journal.pone.0226298

Figure Lengend Snippet: (A) Cell lysate from control siRNA and AP-1 siRNA treated HeLa cells was analyzed by western blotting using anti-γ subunit of AP-1, anti-LZTFL1 and REVERT staining for total protein ( left ). Quantitation of AP-1 level in control siRNA and AP-1 siRNA treated HeLa cells (mean ± SD) is shown. Student’s t-test, n = 3, ***p<0.001 ( right ). (B) Immunofluorescence microscopy of control siRNA and AP-1 siRNA treated HeLa cells fixed and stained with antibodies to γ subunit of AP-1 (green), LZTFL1 (red), p230 (TGN marker, magenta), and DAPI (blue) (top) . Scale bar = 10 μm. Quantitative analysis of relative LZTFL1 intensity at the TGN of control siRNA or AP-1 siRNA treated HeLa cells (mean ± SD) is shown (bottom) . Student’s t-test, n>120 cells, ***p<0.0001.

Article Snippet: Plasmids expressing human LZTFL1 (NM_020347) and TfR1 (NM_003234) with Myc-FLAG tag in pCMV6-Entry vector were purchased from OriGene Technologies (Rockville, MD).

Techniques: Control, Western Blot, Staining, Quantitation Assay, Immunofluorescence, Microscopy, Marker

Journal: PLoS ONE

Article Title: Leucine zipper transcription factor-like 1 binds adaptor protein complex-1 and 2 and participates in trafficking of transferrin receptor 1

doi: 10.1371/journal.pone.0226298

Figure Lengend Snippet: Immunofluorescence microscopy of HeLa cells fixed and stained with antibodies to (A) AP-1 (green), TfR1 (red) and DAPI (blue), scale bar = 10 μm. (B) LZTFL1 (green), TfR1 (red) and DAPI (blue), scale bar = 10 μm. (C) Pearson’s correlation coefficient of the colocalization of AP-1 or LZTFL1 with TfR1 at the TGN (mean ± SD). n>20 cells from three independent experiments. Costes significance test, p = 1. (D) Lysate from HEK293FT cells was immunoprecipitated with mouse IgG and either anti-γ subunit of AP-1 (left) or anti-α subunit of AP-2 (right) conjugated beads. The interacting proteins were eluted by NuPAGE LDS buffer and analyzed by western blotting using anti- γ subunit of AP-1, anti-α subunit of AP-2, anti-TfR1 and REVERT staining for total protein. (E) Lysate from wild-type and LZTFL1 -knockout HeLa cells was immunoprecipitated with anti-LZTFL1-conjugated beads. The LZTFL1-interacting proteins were eluted by NuPAGE LDS buffer and analyzed by western blotting using anti-LZTFL1, anti-TfR1 and REVERT staining for total protein. (F) Factor Xa cleaved MBP-LZTFL1 was incubated with TfR1-Myc-Flag and immunoprecipitated with anti-TfR1 conjugated protein A/G magnetic beads (left) or incubated with purified GST-fused β1 subunits of the AP-1 complex and pulled down with Glutathione Sepharose 4B (right) . The pulled down proteins were analyzed by western blotting using anti-LZTFL1, anti-MBP, anti-TfR1 and anti-GST antibodies. (G) Lysate from HEK293FT cells transiently expressing GFP-FLAG, LZTFL1-FLAG, or the indicated mutants was immunoprecipitated and analyzed by western blotting using anti-FLAG and anti-TfR1 antibodies (left) . Lysate was analyzed by western blotting using anti-FLAG, anti-TfR1, and anti-tubulin antibodies (right) .

Article Snippet: Plasmids expressing human LZTFL1 (NM_020347) and TfR1 (NM_003234) with Myc-FLAG tag in pCMV6-Entry vector were purchased from OriGene Technologies (Rockville, MD).

Techniques: Immunofluorescence, Microscopy, Staining, Immunoprecipitation, Western Blot, Knock-Out, Incubation, Magnetic Beads, Purification, Expressing

Journal: PLoS ONE

Article Title: Leucine zipper transcription factor-like 1 binds adaptor protein complex-1 and 2 and participates in trafficking of transferrin receptor 1

doi: 10.1371/journal.pone.0226298

Figure Lengend Snippet: (A) Wild-type and LZTFL1 -knockout HeLa cells were labeled with 2 mM Sulfo-NHS-LC-Biotin. Biotin-labeled proteins were extracted as described in the Methods and were analyzed by western blotting using anti-TfR1, anti-LZTFL1, and REVERT staining for total protein. (B) Quantitation of cell surface TfR1 level in wild-type and LZTFL1 -knockout HeLa cells (mean ± SD). Student’s t-test, n = 3, ***p<0.0001. (C) Biotin-labeled proteins were extracted from wild-type and LZTFL1 -knockout HeLa cells as described in the Methods and were analyzed by western blotting using anti-EGFR, anti-CI-MPR, anti-LZTFL1, and REVERT staining for total protein. (D) Quantitation of cell surface EGFR and CI-MPR levels in wild-type and LZTFL1-knockout HeLa cells (mean + SD). Student’s t-test, n = 4, n.s., not significant. (E) Wild-type and LZTFL1 -knockout HeLa cells were labeled with 2 mM Sulfo-NHS-SS-Biotin at 4°C. Biotin-labeled cells were incubated with complete DMEM for 0, 10, and 20 minutes to allow protein internalization. The remaining biotin-labeled cell surface proteins were removed with cold 100 mM Mesna followed by quenching with 120 mM iodoacetamide. Biotin-labeled proteins were then extracted as described in the Methods and were analyzed by western blotting using anti-TfR1 antibody, anti-LZTFL1 antibody, and REVERT staining for total protein. (F) Quantitation of internalized TfR1 level in wild-type and LZTFL1 -knockout HeLa cells (mean ± SD). Student’s t-test, n = 4, **p<0.005.

Article Snippet: Plasmids expressing human LZTFL1 (NM_020347) and TfR1 (NM_003234) with Myc-FLAG tag in pCMV6-Entry vector were purchased from OriGene Technologies (Rockville, MD).

Techniques: Knock-Out, Labeling, Western Blot, Staining, Quantitation Assay, Incubation

Journal: PLoS ONE

Article Title: Leucine zipper transcription factor-like 1 binds adaptor protein complex-1 and 2 and participates in trafficking of transferrin receptor 1

doi: 10.1371/journal.pone.0226298

Figure Lengend Snippet: (A) Wild-type and LZTFL1 -knockout HeLa cells were loaded with 25 μg/ml of Tfn-568 on ice for 30 minutes, washed, and incubated with complete DMEM for 0, 20, and 30 minutes at 37°C. Time course of Tfn-568 internalization was monitored by loss of fluorescence. Scale bar = 20 μm. (B) Quantitation of Tfn-568 internalization intensity as a percentage normalized with the intensity at time point 0 (100%) in wild-type and LZTFL1 -knockout HeLa cells (mean ± SD). Student’s t-test, n>100 cells, ***p<0.0001. (C) Wild-type and LZTFL1 -knockout HeLa cells were incubated with 25 μg/ml Tfn-568 for the indicated times, and the Tfn uptake level was measured by the accumulation of Tfn-568. Scale bar = 20 μm. (D) Quantitation of Tfn-568 accumulation in wild-type and LZTFL1 -knockout HeLa cells (mean ± SD). Student’s t-test, n>100 cells, ***p<0.0001. (E) Wild-type and LZTFL1 -knockout HeLa cells were loaded with 25 μg/ml Tfn-568 for 30 minutes, washed, and incubated with complete DMEM for the indicated times. Time course of Tfn-568 efflux was monitored by loss of fluorescence. Scale bar = 20 μm. (F) Quantitation of Tfn-568 efflux intensity as a percentage normalized with the intensity at time point 0 (100%) in wild-type and LZTFL1 -knockout HeLa cells (mean ± SD). Student’s t-test, n>100 cells, ***p<0.0001.

Article Snippet: Plasmids expressing human LZTFL1 (NM_020347) and TfR1 (NM_003234) with Myc-FLAG tag in pCMV6-Entry vector were purchased from OriGene Technologies (Rockville, MD).

Techniques: Knock-Out, Incubation, Fluorescence, Quantitation Assay

Journal: Virology

Article Title: Lipid rafts both in cellular membrane and viral envelope are critical for PRRSV efficient infection.

doi: 10.1016/j.virol.2015.06.005

Figure Lengend Snippet: Fig. 3. Distribution of certain glycoproteins on the cellular lipid raft during PRRSV entry and lipid raft-location of receptor CD163. (A) The distribution of Gp3, Gp4, Gp5, and CD163 in the different sucrose gradient fractions during entry. PAMs were incubated with Hpv at an MOI of 5 at 4 1C for 1 h and then switched to 37 1C for 2 h. Cells were extracted in Triton X-100 and fractionated by discontinuous sucrose gradient centrifugation as described in Materials and Methods. The fractions were separated by SDS-12% PAGE and transferred to PVDF membranes for immunoblot analysis for nonraft marker CD71, raft maker caveolin (Cav), Gp3, Gp4, Gp5, or CD163. (B) The distribution of Gp3, Gp4, Gp5, and CD163 in different sucrose gradient fractions during PRRSV entry upon MβCD pretreatment. PAMs were pretreated with 20 mM MβCD for 1 h and then incubated with Hpv at an MOI of 5 at 4 1C for 1 h and then switched to 37 1C for 2 h. Cells were extracted in Triton X-100 and fractionated by discontinuous sucrose gradient centrifugation as described in Materials and Methods. The fractions were separated by SDS-12% PAGE and transferred to PVDF membranes for immunoblot analysis for CD71, Cav, Gp3, Gp4, Gp5, or CD163. (C) MβCD treatment affected the distribution of CD163. Hela cells were transfected with plasmid encoding CD163 and treated with 20 mM MβCD at 37 1C for 1 h at 24 h post transfection. The cells treated with PBS were set as a control. The expression of CD163 on cell membrane was stained by indirect immunofluorescence assay after MβCD treatment. (D) Association of CD163 with lipid rafts. PAMs were extracted in Triton X-100 and fractionated on sucrose gradients. Proteins were resolved on SDS-PAGE and transferred to PVDF membrane for immunoblot analysis of CD163, Cav, and CD71.

Article Snippet: Anti-CD71 antibody was purchased from Acris Antibodies GmbH.

Techniques: Incubation, Gradient Centrifugation, Western Blot, Marker, Transfection, Plasmid Preparation, Control, Expressing, Membrane, Staining, SDS Page

Journal: Virology

Article Title: Lipid rafts both in cellular membrane and viral envelope are critical for PRRSV efficient infection.

doi: 10.1016/j.virol.2015.06.005

Figure Lengend Snippet: Fig. 4. Association of lipid raft with PRRSV replication. (A) The effect of PRRSV replication at the indicated time intervals in PAMs upon MβCD treatment. PAMs were infected with Hpv at an MOI of 1 for 12 h (represented as 0 h on graph), and then the medium was replaced with fresh medium containing 10 mM MβCD. ORF7 RNA level in cells was analyzed using real-time RT-PCR at 0, 6, and 12 h after treatment. Data shown as mean7standard deviation from three independent experiments. Significant differences compared with 0 h group are denoted by * (Po0.05) and ** (Po0.01). (B) Association of Nsp9 with raft. PAMs were harvested 24 h after infection with Hpv at an MOI of 1. Cells were extracted in Triton X-100 and fractionated by discontinuous sucrose gradient centrifugation as described in Materials and Methods. Each fraction was resolved on SDS-PAGE and transferred to PVDF membrane for immunoblot analysis of Nsp9, Cav, and CD71. (C) PAMs were harvested 24 h after infection with Hpv at an MOI of 1. Cells were treated with 20 mM MβCD for 1 h and then extracted in Triton X-100 and fractionated by discontinuous sucrose gradient centrifugation as described in Materials and Methods. Each fraction was resolved on SDS-PAGE and transferred to PVDF membrane for immunoblot analysis of Nsp9, Cav, and CD71.

Article Snippet: Anti-CD71 antibody was purchased from Acris Antibodies GmbH.

Techniques: Infection, Quantitative RT-PCR, Gradient Centrifugation, SDS Page, Membrane, Western Blot

Journal: Virology

Article Title: CD4- and dynamin-dependent endocytosis of HIV-1 into plasmacytoid dendritic cells.

doi: 10.1016/j.virol.2011.11.026

Figure Lengend Snippet: Fig. 6. Intracellular trafficking of HIV-1 captured by plasmacytoid dendritic cells (PDC). PDC were exposed to HIV-1NL4-3 at 4 °C for 30 min and then incubated at 37 °C for different time periods. Cells were labeled using a biotinylated antibody to the viral p24 capsid antigen (red), and monoclonal antibodies (green) to (A) the Rab GTPases 5, 7, and 9, (B) the tetraspanins CD63 and CD81, and the marker protein for recycling endosomes, CD71. Cell nuclei were stained using DAPI (blue). For quantification, double-positive cells were counted and the proportion of cells with a colocalization event was calculated. Data represent three independent confocal experiments, evaluating a total of 366, 412, and 304 double-positive cells for Rab5/7/9, and 368, 197 and 192 double-positive cells for CD63, CD81 or CD71. Data summarize all time points and are given as mean and standard error. Details are provided in Fig. S2. ***pb0.001.

Article Snippet: The biotinylated goat anti-p24 antibody (1:100) (ViroStat, Portland, ME) was incubated at 4 °C overnight together with the respective primary antibodies, the murine antibodies against LAMP-1 (see above; 1:100), CD71 (Acris Antibodies, Herford, Germany; 1:100), CD63 (1:1000) and CD81 (1:1000) (BD Biosciences, Heidelberg, Germany), or rabbit antibodies against EEA-1 (1:100), the Rab GTPases 5 (1:100), 7 (1:50), and 9 (1:100), Clathrin (1:50), and Caveolin-1 (1:400) (Cell Signaling, distributed by New England Biolabs, Frankfurt, Germany).

Techniques: Incubation, Labeling, Bioprocessing, Marker, Staining

Journal: Virology

Article Title: CD4- and dynamin-dependent endocytosis of HIV-1 into plasmacytoid dendritic cells.

doi: 10.1016/j.virol.2011.11.026

Figure Lengend Snippet: Fig. 7. Characterization of entry mechanism of HIV-1 into PDC. (A) PDC were exposed to HIV-1NL4-3 at 4 °C for 30 min and then incubated at 37 °C for different time periods. Cells were stained using a biotinylated antibody to the viral p24 capsid antigen (red) and monoclonal antibodies against clathrin or caveolin-1 (green). For quantification, double-positive cells were counted and the proportion of cells with a colocalization event was calculated. Cell nuclei were stained using DAPI (blue). Data represent three independent confocal experiments, evaluating a total of 111 and 152 double-positive cells for clathrin and caveolin-1, respectively. The results summarize all time points and are given as mean and standard error. *pb0.05. As a control, HIV-1 exposed PDC of two different donors were stained with antibodies against clathrin (green) and CD71 (red). Results are representative for 172, 282, and 180 double-positive cells at 5, 15, and 30 min of in- cubation, respectively. (B) PDC were preincubated with mock (media), anti-CD4, human IgG (isotype for anti-CD4), anti-BDCA2, or goat IgG (isotype for BDCA2) for 2 h at 37 °C before they were exposed to HIV-1NL4-3 at 37 °C for 45 min. Cells were labeled with DAPI (blue), anti-p24 (red) and anti-EEA1 (green) and subsequently analyzed by confocal microscopy. The upper and lower panels show endosomal and cytosolic viral particles, respectively. The results are representative of two independent experiments.

Article Snippet: The biotinylated goat anti-p24 antibody (1:100) (ViroStat, Portland, ME) was incubated at 4 °C overnight together with the respective primary antibodies, the murine antibodies against LAMP-1 (see above; 1:100), CD71 (Acris Antibodies, Herford, Germany; 1:100), CD63 (1:1000) and CD81 (1:1000) (BD Biosciences, Heidelberg, Germany), or rabbit antibodies against EEA-1 (1:100), the Rab GTPases 5 (1:100), 7 (1:50), and 9 (1:100), Clathrin (1:50), and Caveolin-1 (1:400) (Cell Signaling, distributed by New England Biolabs, Frankfurt, Germany).

Techniques: Incubation, Staining, Bioprocessing, Control, Labeling, Confocal Microscopy