rabbit anti lysosomal associated membrane protein 2 lamp2 monoclonal antiserum  (Cell Signaling Technology Inc)

Journal: International Journal of Molecular Sciences

Article Title: Downregulation of Protease Cathepsin D and Upregulation of Pathologic α-Synuclein Mediate Paucity of DNAJC6-Induced Degeneration of Dopaminergic Neurons

doi: 10.3390/ijms25126711

Figure Lengend Snippet: DNAJC6 deficiency causes downregulated expression of clathrin heavy chain, LAMP1, LAMP2 or cathepsin D and upregulated expression of p62/SQSTM1 in dopaminergic neurons. After transfection of shRNAs targeting DNAJC6 for 3 days, the protein level of cytosolic clathrin heavy chain was significantly decreased in dopaminergic neurons. Cytosolic protein levels of lysosomal marker proteins LAMP1 or LAMP2 and lysosomal aspartic protease cathepsin D were downregulated in DNAJC6 shRNA-transfected dopaminergic neurons. The protein level of cytosolic autophagy receptor p62/SQSTM1 was upregulated in dopaminergic neurons transfected with shRNAs targeting DNAJC6. Each bar shows the mean ± S.E. value of 6 experiments. ** p < 0.01 compared with control dopaminergic neurons.

Article Snippet: PVDF membranes were then incubated with one of the following diluted primary antiserums purchased from Cell Signaling Technology: (1) rabbit monoclonal anti-clathrin heavy chain antibody; (2) rabbit anti-lysosomal-associated membrane protein 1 (LAMP1) monoclonal antiserum; (3) rabbit monoclonal anti-lysosomal-associated membrane protein 2 (LAMP2) antibody; (4) anti-p62/SQSTM1 rabbit monoclonal antiserum; (5) rabbit polyclonal anti-cathepsin D antibody.

Techniques: Expressing, Transfection, Marker, shRNA, Control

Journal: International Journal of Molecular Sciences

Article Title: Downregulation of Protease Cathepsin D and Upregulation of Pathologic α-Synuclein Mediate Paucity of DNAJC6-Induced Degeneration of Dopaminergic Neurons

doi: 10.3390/ijms25126711

Figure Lengend Snippet: Paucity of DNAJC6 decreases LysoTracker staining intensity of lysosomes and immunofluorescence staining intensity of LAMP2-positive lysosomes in dopaminergic neurons. ( A ) Live cell imaging of lysosomes was conducted by treating differentiated SH-SY5Y dopaminergic neurons with 100 nM LysoTracker Yellow HCK-123, which stains acidic compartments and visualizes lysosomes, for 1 h. A three-day transfection of shRNA1 or shRNA2 targeting DNAJC6 significantly decreased the fluorescence intensity of LysoTracker Yellow staining in dopaminergic neurons. SC shRNA failed to affect lysosomal staining of LysoTracker Yellow HCK-123. Scale bar is 50 μm. ( B ) Immunofluorescence staining of lysosomal marker protein LAMP2 was performed to visualize lysosomes of dopaminergic neurons. Following a 3-day transfection of shRNAs targeting DNAJC6, the fluorescence intensity of LAMP2-positive puncta was significantly decreased in dopaminergic neurons. SC shRNA did not affect the immunofluorescence staining intensity of LAMP2. Scale bar is 60 μm. Each bar shows the mean ± S.E. value of 6 experiments. ** p < 0.01 compared with control dopaminergic neurons.

Article Snippet: PVDF membranes were then incubated with one of the following diluted primary antiserums purchased from Cell Signaling Technology: (1) rabbit monoclonal anti-clathrin heavy chain antibody; (2) rabbit anti-lysosomal-associated membrane protein 1 (LAMP1) monoclonal antiserum; (3) rabbit monoclonal anti-lysosomal-associated membrane protein 2 (LAMP2) antibody; (4) anti-p62/SQSTM1 rabbit monoclonal antiserum; (5) rabbit polyclonal anti-cathepsin D antibody.

Techniques: Staining, Immunofluorescence, Live Cell Imaging, Transfection, Fluorescence, shRNA, Marker, Control

Journal: PLOS Pathogens

Article Title: The HN protein of Newcastle disease virus induces cell apoptosis through the induction of lysosomal membrane permeabilization

doi: 10.1371/journal.ppat.1011981

Figure Lengend Snippet: (A&B) HeLa cells infected with Herts/33 at 0.01 MOI for indicated timepoints. The mRNA levels (A) and fluorescence images (B) of LAMP1 and LAMP2 were measured by RT-qPCR or observed by confocal microscopy, respectively. The mRNA levels of LAMP1 and LAMP2 were normalized to GAPDH using the 2 −ΔΔCt method. Scale bars, 20 μm. (C-E) HeLa cells infected with Herts/33 at 0.01 MOI for indicated time points or infected with Herts/33 at indicated MOIs for 24h. The protein levels of LAMP1 and LAMP2 (C) or LAMP3, LIMPII, and LAPTM5 (E) were assessed by Western blotting. The deglycosylated LAMP1 and LAMP2 in PNGase F-treated cell lysates were analyzed by Western blotting (E). (F-H) HeLa cells were infected with Herts/33 at 0.01 MOI for 12h or mock-infected. The remaining amounts of LAMP1 and LAMP2 after treatment with CHX (20 μg/mL) for indicated time points were measured using Western blotting (F). The remaining amount of LAMP1 (G) and LAMP2 (H) was calculated as the fold change of the protein present at 0h. (I-K) NDV-infected (0.01 MOI, 6h) HeLa cells were co-treated with CHX (20 μg/mL) and NH4CL (50 mM), or Ca-074 (10 μM), or Pep A (10 μM), or 3-MA (5mM), or MG132 (5 μM) or mock-treated for 12h. LAMP1 and LAMP2 protein levels in cell lysates were analyzed by Western blotting (I). The remaining amounts of LAMP1 (J) and LAMP2 (K) were calculated as the fold change of the protein to mock-treated cells at 0h. All quantitative data represent means ± SD (n = 3). Significance was assessed using Two-way ANOVA with Dunnett’s multiple comparisons test (A), or unpaired two-tailed Student’s t-test (G&H), or One-way ANOVA with Dunnett’s multiple comparisons test (J&K).

Article Snippet: Goat anti-rabbit IgG H&L (Alexa Fluor 647) secondary antibody (ab150083), goat anti-mouse IgG H&L (Alexa Fluor 488) secondary antibody (ab150117), anti-LAMP2 rabbit monoclonal antibody (ab13524) and anti-Cleaved Caspase 7 rabbit monoclonal antibody (ab256469) were purchased from Abcam, Waltham, USA.

Techniques: Infection, Fluorescence, Quantitative RT-PCR, Confocal Microscopy, Western Blot, Two Tailed Test

Journal: PLOS Pathogens

Article Title: The HN protein of Newcastle disease virus induces cell apoptosis through the induction of lysosomal membrane permeabilization

doi: 10.1371/journal.ppat.1011981

Figure Lengend Snippet: (A) Western blotting analysis of LAMP1 and LAMP2 protein levels in HeLa cells after transfection with plasmids expressing indicated viral proteins for 48h. (B&C) Western blotting analysis of LAMP1 and LAMP2 protein levels in HeLa cells after transfection with indicated doses of plasmids expressing HN (B) or F (C) protein for 48h. (D) Confocal microscopy images of LMP level in HeLa cells using anti-galectin 3 (green) and anti-LAMP1 (red) antibodies after transfection with indicated plasmids for 48h. Scale bars, 20 μm. (E&F) The interaction between HN and LAMP1 (E) or LAMP2 (F) was detected by an immunoprecipitation assay with anti-flag or control IgG antibodies after transfection with indicated plasmids into HeLa cells for 48h. (G&H) Confocal microscopy images of the interaction between HN and LAMP1 (G) or LAMP2 (H) using anti-HN, anti-LAMP1, or anti-LAMP2 antibodies after infection of HeLa cells with Herts/33 at 0.01 MOI or mock-infected for 24h. Scale bars, 20 μm. (K) HeLa cells were transfected with siRNAs targeting LAMP1 or LAMP2 or plasmids expressing LAMP1 or LAMP2 for 48h. Then LAMP1 or LAMP2 protein levels were detected by Western blotting. The red boxes indicate the siRNA selected for this study. (I&J) HeLa cells were transfected with plasmids expressing LAMP1 or LAMP2 (M) or siRNAs targeting LAMP1 or LAMP2 (N) for 48h. Then the LMP level was observed by confocal microscopy using anti-galectin 3 (green) and anti-LAMP1 (red) antibodies following transfection with plasmid expressing HN protein for another 48h. Scale bars, 20 μm. (L&M) Manders’ Colocalization Coefficients of galectin 3 with LAMP1 are quantified by ImageJ software and shown in (L) for (I) and (M) for (J). (N&O) HeLa cells were transfected with plasmids expressing LAMP1 or LAMP2 (N) or siRNAs targeting LAMP1 or LAMP2 (O) for 48h. Then the apoptosis rate was measured by AnnexinV-FITC/PI staining using flow cytometry following transfection with plasmid expressing HN protein for 48h. Error bars are SDs for 15 cells (L&M), or SDs for a triplicate analysis of three independent experiments (N&O). All significance analyses were assessed using One-way ANOVA with Dunnett’s multiple comparisons test.

Article Snippet: Goat anti-rabbit IgG H&L (Alexa Fluor 647) secondary antibody (ab150083), goat anti-mouse IgG H&L (Alexa Fluor 488) secondary antibody (ab150117), anti-LAMP2 rabbit monoclonal antibody (ab13524) and anti-Cleaved Caspase 7 rabbit monoclonal antibody (ab256469) were purchased from Abcam, Waltham, USA.

Techniques: Western Blot, Transfection, Expressing, Confocal Microscopy, Immunoprecipitation, Infection, Plasmid Preparation, Software, Staining, Flow Cytometry

Journal: PLOS Pathogens

Article Title: The HN protein of Newcastle disease virus induces cell apoptosis through the induction of lysosomal membrane permeabilization

doi: 10.1371/journal.ppat.1011981

Figure Lengend Snippet: (A) Western blotting analysis of LAMP1 and LAMP2 deglycosylation and degradation in HeLa cells transfected with plasmid expressing HN protein and treated with Peramivir (30 μg/mL) or Zanamivir (20 μg/mL) at the indicated time points post-transfection. (B&C) HeLa cells were transfected with plasmids expressing HN protein or mock-transfected. Then the cells were treated with Peramivir (30 μg/mL) or Zanamivir (20 μg/mL) or DMSO or untreated for 48h. The LMP level was observed by confocal microscopy using anti-galectin 3 (green) and anti-LAMP1 (red) antibodies. Scale bars, 20 μm (B). Manders’ Colocalization Coefficients of galectin 3 with LAMP1 are quantified by ImageJ software and shown in (C). (G) Sialidase activity was measured in HeLa cells transfected with the indicated plasmids for 48h and is presented as relative change to the cells transfected with plasmids expressing wild-type HN. The red dotted line indicates the difference threshold. (F) Western blotting analysis of LAMP1 and LAMP2 deglycosylation and degradation in HeLa cells transfected with the indicated plasmids or mock-transfected for 48h. (D&E) The interaction between HN and LAMP1 (D) or LAMP2 (E) in HeLa cells was detected by an immunoprecipitation assay with anti-flag or control IgG antibodies after transfection with indicated plasmids for 48h. (H) The LMP level was observed by confocal microscopy using anti-galectin 3 (green) and anti-LAMP1 (red) antibodies following transfection with the indicated plasmids or mock-transfected for 48h. Scale bars, 20 μm. (I) Manders’ Colocalization Coefficients of galectin 3 with LAMP1 are calculated by ImageJ software. (J) The apoptosis rate was measured by AnnexinV-FITC/PI staining using flow cytometry following transfection with indicated plasmids or mock-transfection for 48h. Error bars are SDs for a triplicate analysis of three independent experiments (G&J), or SDs for 15 cells (C&H). All significance analyses were assessed using One-way ANOVA with Dunnett’s multiple comparisons test.

Article Snippet: Goat anti-rabbit IgG H&L (Alexa Fluor 647) secondary antibody (ab150083), goat anti-mouse IgG H&L (Alexa Fluor 488) secondary antibody (ab150117), anti-LAMP2 rabbit monoclonal antibody (ab13524) and anti-Cleaved Caspase 7 rabbit monoclonal antibody (ab256469) were purchased from Abcam, Waltham, USA.

Techniques: Western Blot, Transfection, Plasmid Preparation, Expressing, Confocal Microscopy, Software, Activity Assay, Immunoprecipitation, Staining, Flow Cytometry

Journal: PLOS Pathogens

Article Title: The HN protein of Newcastle disease virus induces cell apoptosis through the induction of lysosomal membrane permeabilization

doi: 10.1371/journal.ppat.1011981

Figure Lengend Snippet: NDV enters host cells and utilizes its genome for transcription and translation of the HN protein. The HN protein digests the sialic acid at the end of the glycan chains of LAMP1 and LAMP2 via its sialidase activity, leading to desialylation. The desialylated LAMP1 and LAMP2 undergo deglycosylation and degradation in the lysosome by CTSB, leading to LMP and the leakage of CTSB and CTSD. Consequently, these translocated CTSB and CTSD promote the degradation of Bcl-2, the cleavage of Bid into tBid, and the mitochondrial translocation of Bax, thereby inducing MOMP. This process further exacerbates LMP by inducing ROS release while simultaneously activating mitochondria-dependent apoptosis through the release of Cyt C.

Article Snippet: Goat anti-rabbit IgG H&L (Alexa Fluor 647) secondary antibody (ab150083), goat anti-mouse IgG H&L (Alexa Fluor 488) secondary antibody (ab150117), anti-LAMP2 rabbit monoclonal antibody (ab13524) and anti-Cleaved Caspase 7 rabbit monoclonal antibody (ab256469) were purchased from Abcam, Waltham, USA.

Techniques: Activity Assay, Translocation Assay