antibody against human lamp2  (Developmental Studies Hybridoma Bank)

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Hydroxynonenal Causes Hepatocyte Death by Disrupting Lysosomal Integrity in Nonalcoholic Steatohepatitis

doi: 10.1016/j.jcmgh.2022.06.008

Figure Lengend Snippet: Induction of lysosomal membrane permeabilization/rupture in hepatocytes by HNE. A , Changes in lysosomes of HepG2 cells after the addition of EPI and HNE were observed by time-lapse imaging using LysoTracker. Blue , Hoechst; red , LysoTracker. Square on the top right indicates morphological changes that were observed on bright field imaging. B , Electron microscopy images of HepG2 cells. Black squares show magnified images of membrane-bound lysosomes in the control (Cont) and disruption of the lysosomal limiting membrane in HNE-treated HepG2 cells (HNE), respectively. L, lysosome. C , HepG2 cells were treated with 25 μM HNE, and imaged 0, 2, and 6 hours later. Blue , DAPI; green , CTSB; red , LysoTracker; yellow , merge. D , Area occupied by LysoTracker and CTSB for each particle in HepG2 cells at a 20× image. The analysis was performed with 10 images in each group. E , Images show immunoreactivity of HepG2 cells for CTSB and LAMP2 before (0) and 6 hours after the treatment of 25 μM HNE. Blue , DAPI; green , CTSB; red , LAMP2; yellow , merge. F , Electron microscopy images of Huh-7. Black squares show magnified images of membrane-bound lysosomes in the control (Cont) and disruption of the lysosomal limiting membrane in HNE-treated Huh7 cells (HNE), respectively. L, lysosome. G , Huh-7 cells were treated with 25 μM HNE, and imaged 0, 2, and 6 hours later. Blue , DAPI; green , CTSB; red , LysoTracker; yellow , merge. H , Area occupied by LysoTracker and CTSB for each particle in Huh7 cells at a 20× image of Huh7. I , Images show immunoreactivity of Huh-7 cells for CTSB and LAMP2 before (0) and 6 hours after the treatment of 25 μM HNE. Blue , DAPI; green , CTSB; red , LAMP2; yellow , merge.

Article Snippet: The following primary antibodies for LAMP2, CTSB, μ-calpain, and were used: a mouse monoclonal antibody against African green monkey LAMP2 (ab25631, Abcam, Cambridge, UK), a rabbit monoclonal antibody against human CTSB (D1C7Y, Cell Signaling, Danvers, MA), a rabbit antiserum anti-human activated μ-calpain antibody (order made by PEPTIDE Institute, Ibaraki, Osaka, Japan) that binds with the activated (76 kDa), not inactivated (80 kDa) form of μ-calpain.

Techniques: Membrane, Imaging, Electron Microscopy, Control, Disruption

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Hydroxynonenal Causes Hepatocyte Death by Disrupting Lysosomal Integrity in Nonalcoholic Steatohepatitis

doi: 10.1016/j.jcmgh.2022.06.008

Figure Lengend Snippet: Lysosomal membrane permeabilization/rupture in hepatocytes of patients with NASH. A , Immunofluorescence staining of liver tissue from patients with nonfatty liver disease and NASH. Blue , DAPI; green , CTSB; red , LAMP2. The area highlighted in the yellow square is a magnified image of the nonfatty liver, whereas the red square is a magnified image of the NASH liver. B , Relationship between the HNE staining score (patient numbers; grade 0, n = 5; grade 1, n = 9; grade 2, n = 12) and granule sizes for LAMP2. The area of particles stained with LAMP2 in 20× image was calculated, respectively. C , Electron microscopy images of the non-fatty liver and NASH liver. Lysosomes with clear limiting membrane structures were observed in the nonfatty liver ( white arrowheads ). In contrast, lysosomes in the NASH liver showed disintegrity ( yellow arrowheads ). Furthermore, in some lysosomes, the lysosomal membrane was disrupted, and contents leaked out ( yellow arrow ). D , A Western blotting analysis of μ-calpain in the livers of 5 patients with nonfatty liver (normal) and 5 patients with NASH is shown. P, protein marker. E , Bands of panel D are quantified and shown as relative fold ratios.

Article Snippet: The following primary antibodies for LAMP2, CTSB, μ-calpain, and were used: a mouse monoclonal antibody against African green monkey LAMP2 (ab25631, Abcam, Cambridge, UK), a rabbit monoclonal antibody against human CTSB (D1C7Y, Cell Signaling, Danvers, MA), a rabbit antiserum anti-human activated μ-calpain antibody (order made by PEPTIDE Institute, Ibaraki, Osaka, Japan) that binds with the activated (76 kDa), not inactivated (80 kDa) form of μ-calpain.

Techniques: Membrane, Immunofluorescence, Staining, Electron Microscopy, Western Blot, Marker

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Hydroxynonenal Causes Hepatocyte Death by Disrupting Lysosomal Integrity in Nonalcoholic Steatohepatitis

doi: 10.1016/j.jcmgh.2022.06.008

Figure Lengend Snippet: HNE induces lysosomal disintegrity by activating μ -calpain in hepatocytes of Japanese macaque monkeys. A , Macroscopic findings of livers in the control (Cont) and in monkeys treated with HNE (HNE). Black arrows show regional discoloration. B , Hematoxylin and eosin staining and HNE immunostaining of liver tissue from the control group (Cont) and HNE-treated group (HNE). HNE immunoreactivity was observed in hepatocytes. C , The expression of liver HNE protein adducts in the control (Cont) and in monkeys treated with HNE (HNE) was evaluated by a Western blotting analysis. In the Western blotting analysis, HNE is depicted as HNE protein adducts of various molecular weights. P, protein marker. D , Bands of panel C are quantified and shown as relative fold ratios. E , Alterations in ALT levels before the HNE treatment and increases after the HNE treatment are shown. F , Comparison of immunofluorescence staining in the control group (Cont) and HNE-treated group (HNE). Blue , DAPI; green , CTSB; red , LAMP2; yellow , merge. The yellow square shows a magnified image of the liver in the control group, whereas the red square shows a magnified image of the liver in the HNE-treated group. G , The area of particles stained with LAMP2 is shown in 20× image for each monkey. H , The number of stained areas that were 10 μm 2 or larger in panel G is shown. I , Electron microscopy images of livers in the control (Cont) and HNE-treated groups (HNE). Black squares show magnified images of membrane-bound lysosomes in the control (Cont) and disruption of the lysosomal limiting membrane in the HNE-treated group (HNE). L, lysosome. J , A Western blotting analysis showing the expression of activated μ-calpain in the livers of the control (Cont) and HNE-treated groups (HNE). P, protein marker. K , Bands of panel J are quantified and shown as relative fold ratios.

Article Snippet: The following primary antibodies for LAMP2, CTSB, μ-calpain, and were used: a mouse monoclonal antibody against African green monkey LAMP2 (ab25631, Abcam, Cambridge, UK), a rabbit monoclonal antibody against human CTSB (D1C7Y, Cell Signaling, Danvers, MA), a rabbit antiserum anti-human activated μ-calpain antibody (order made by PEPTIDE Institute, Ibaraki, Osaka, Japan) that binds with the activated (76 kDa), not inactivated (80 kDa) form of μ-calpain.

Techniques: Control, Staining, Immunostaining, Expressing, Western Blot, Marker, Comparison, Immunofluorescence, Electron Microscopy, Membrane, Disruption

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Hydroxynonenal Causes Hepatocyte Death by Disrupting Lysosomal Integrity in Nonalcoholic Steatohepatitis

doi: 10.1016/j.jcmgh.2022.06.008

Figure Lengend Snippet: Administration of Alda-1 in CDAA mice suppresses liver fibrosis. A , Hematoxylin and eosin staining and HNE immunostaining of liver tissue from the control mice fed the standard diet (Cont), CDAA mice (CDAA) and Alda-1-treated CDAA mice (CDAA+Alda-1) for 8 weeks. B , The expressions of liver HNE protein adducts in CDAA mice (CDAA) and CDAA mice with Alda-1 treatment (CDAA+Alda-1) for 4 (4w) and 8 (8w) weeks were evaluated by a Western blotting analysis. C , Bands of panel B are quantified and shown as relative fold ratios. D , Comparison of histopathological scores of CDAA mice (CDAA) and Alda-1 treated CDAA mice (CDAA+Alda-1) for 8 weeks, such as steatosis score, lobular inflammation score (Lobular inf), ballooning score, and NAFLD activity score (NAS) is shown. E , Sirius red staining of liver tissue from CDAA mice (CDAA) and Alda-1-treated CDAA mice (CDAA+Alda-1) for 8 weeks. F , Area occupied by Sirius red staining in a 20× image were compared between the CDAA mice (CDAA) and Alda-1 treated CDAA mice (CDAA+Alda-1) for 8 weeks. The area was calculated for each of the 10 images and averaged. G , Real-time PCR analysis of IL-1β, IL-6, tumor necrosis factor (TNF), and toll-like receptor (TRL)-4 expression in CDAA mice ( blue circle ) and Alda-1 treated CDAA mice ( red triangle ) for 4 and 8 weeks. H , Immunofluorescence staining of livers in control mice (Cont) and the CDAA mice (CDAA). Blue , DAPI; green , CTSB; red , LAMP2. I , Electron microscopy images of liver tissue collected from control mice (Cont) and CDAA mice (CDAA) for 8 weeks. The black arrow shows the disruption of the lysosomal limiting membrane. L, lysosome. J , Immunofluorescence LAMP2 staining of liver tissue from CDAA mice (CDAA) and Alda-1-treated CDAA mice (CDAA+Alda-1) for 8 weeks. K , The area of particles stained with LAMP2 in CDAA mice (CDAA) and Alda-1 treated CDAA mice (CDAA+Alda-1) for 4 weeks is shown. L , The number of stained areas that were 10 μm 2 or larger in panel K is shown. M , The area of particles stained with LAMP2 in CDAA mice (CDAA) and Alda-1 treated CDAA mice (CDAA+Alda-1) for 8 weeks is shown. N , The number of stained areas that were 10 μm 2 or larger in panel M is shown.

Article Snippet: The following primary antibodies for LAMP2, CTSB, μ-calpain, and were used: a mouse monoclonal antibody against African green monkey LAMP2 (ab25631, Abcam, Cambridge, UK), a rabbit monoclonal antibody against human CTSB (D1C7Y, Cell Signaling, Danvers, MA), a rabbit antiserum anti-human activated μ-calpain antibody (order made by PEPTIDE Institute, Ibaraki, Osaka, Japan) that binds with the activated (76 kDa), not inactivated (80 kDa) form of μ-calpain.

Techniques: Staining, Immunostaining, Control, Western Blot, Comparison, Activity Assay, Real-time Polymerase Chain Reaction, Expressing, Immunofluorescence, Electron Microscopy, Disruption, Membrane