Anti-Lysozyme LYZ Rabbit Monoclonal Antibody

Western blot analysis of Lysozyme using anti-Lysozyme antibody (M01811).
Electrophoresis was performed on a 5-20% SDS-PAGE gel at 70V (Stacking gel) / 90V (Resolving gel) for 2-3 hours. The sample well of each lane was loaded with 30 ug of sample under reducing conditions.
Lane 1: human HL-60 whole cell lysates,
Lane 2: human U937 whole cell lysates,
Lane 3: mouse RAW264.7 whole cell lysates.
After electrophoresis, proteins were transferred to a nitrocellulose membrane at 150 mA for 50-90 minutes. Blocked the membrane with 5% non-fat milk/TBS for 1.5 hour at RT. The membrane was incubated with rabbit anti-Lysozyme antigen affinity purified monoclonal antibody (Catalog # M01811) at 1:5000 overnight at 4°C, then washed with TBS-0.1%Tween 3 times with 5 minutes each and probed with a goat anti-rabbit IgG-HRP secondary antibody at a dilution of 1:5000 for 1.5 hour at RT. The signal is developed using an Enhanced Chemiluminescent detection (ECL) kit (Catalog # EK1002) with Tanon 5200 system. A specific band was detected for Lysozyme at approximately 15 kDa. The expected band size for Lysozyme is at 17 kDa.

IF analysis of Lysozyme using anti-Lysozyme antibody (M01811). Lysozyme was detected in a paraffin-embedded section of mouse colon tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated at 1:50 rabbit anti-Lysozyme Antibody (M01811) overnight at 4°C. DyLight®488 Conjugated Goat Anti-Rabbit IgG (BA1127) was used as secondary antibody at 1:500 dilution and incubated for 30 minutes at 37°C. The section was counterstained with DAPI. Visualize using a fluorescence microscope and filter sets appropriate for the label used.

MG53 attenuates intestinal injury in mouse models. a Serum IL-6 and IL-18 levels of MG53-TG and their WT littermates at the indicated time points following DSS treatment. n = 3 for each group. Overexpression of MG53 attenuated DSS-induced IBD symptoms, including disease activity index (DAI; b ), body weight change ( c ), disruption of intestinal structure as demonstrated by H&E staining of the jejunum, ileum, and colon ( d ; scale bar, 100 μm), and shortening of the colon as evidenced by the representative images ( e ) and statistic results of colon length ( f ) in MG53-TG and WT mice. n = 7 for each group. g , h Depletion of MG53 exacerbated DSS-induced IBD symptoms. The DAI ( g ) and body weight change ( h ) of MG53-KO and the WT littermates at the indicated time points after DSS challenge. n = 5 for each group. Immunofluorescence staining of Ki-67 (red) to indicate the TA zone and the statistic results of its length in MG53-TG ( i , n = 20 for each group) or MG53-KO ( j , n = 13 for each group) as compared with their corresponding WT littermates. The nuclei were indicated by DAPI staining (blue); scale bar, 100 μm. Normal distribution was confirmed by Shapiro–Wilk test. Data were analyzed using two tailed paired t test ( a – c , f – j ). All data were presented as mean ± s.e.m. * p < 0.05, ** p < 0.01, and *** p < 0.001 as compared with the corresponding controls Full size image
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PPARα mediates the effect of MG53 in promoting asymmetric division of Lgr5 + cells. Representative images ( a ) and statistic results ( b , n = 3 for each group) of asymmetric division of Lgr5 + cells with or without GW6471 (10 μM, 24 h) treatment. Lgr5 + cells were in green; all the cells were visualized by tubulin (red). Scale bar, 10 μm. c FACS analysis of Lgr5 + , Atoh1 + , and Notch1 + cells in the Lgr5 and MG53;Lgr5 organoids treated with GW6471 or vehicle. d Immunofluorescence staining of Ki-67 (red) and PPARα (green) and statistic results of PPARα positive cells ( n = 3 for each group) and the length of TA zone ( n = 18 for each group) in mice treated GW6471 or vehicle. Nuclei were stained with DAPI (blue); scale bar, 100 μm. Representative images and statistic results of PAS staining of goblet cells in the ileum ( e ; n = 10 for each group; TG, MG53-TG; scale bar, 50 μm) and colon ( f ; n = 10 for each group; scale bar, 100 μm). Normal distribution was confirmed by Shapiro–Wilk test. Data were analyzed using one-way ANOVA with Tukey post hoc test ( b , d , e , and f ) and were presented as mean ± s.e.m. ns not significant and *** p < 0.001 as compared with the corresponding controls Full size image
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MG53 overexpression promotes secretory lineage commitment. a Immunofluorescence staining of BrdU (red) and EdU (yellow) in the ileum of MG53-TG and their WT littermates subjected to DSS treatment and administered BrdU 48 h and EdU 24 h before euthanasia. n = 10 for each group. Scale bar, 100 μm. b UMAP of scRNA-seq of Epcam + CD45 − intestinal epithelial cells collected from MG53-TG and their WT littermates on Day 7 of DSS treatment, n = 5 for each group. c Proportions of stem, secretory, and absorptive cells in general and each particular cell clusters. d Representative images and statistic results of PAS staining of goblet cells of MG53-TG and WT mice in the colon. n = 10 for each group. Scale bar, 100 μm. e Representative images of intestinal organoids derived from MG53;Lgr5 mice and their Lgr5 littermates at the indicated time points. f Relative mRNA levels of the marker genes in the transcriptome analysis of the crypt organoids. n = 4 for each group. g Representative images of colonic organoids derived from MG53;Lgr5 mice and their Lgr5 littermates at the indicated time points. h Representative FACS profiles and statistic results of Lgr5 + cells in the colonic organoids derived from MG53;Lgr5 and the control Lgr5 mice. n = 6 for each group. Normal distribution was confirmed by Shapiro–Wilk test. Data were analyzed using two tailed paired t test ( a , d , f , and h ), and were presented as mean ± s.e.m. * p < 0.05, ** p < 0.01, and *** p < 0.001 as compared with the corresponding controls Full size image
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PPAR signaling is enhanced by MG53 overexpression. a Trajectory analysis of Epcam + CD45 − intestinal epithelial cells Using Monocle 2. b KEGG pathway enrichment analysis of the differentially expressed genes between the secretory and absorptive branches. GSEA results ( c ) and the heatmap of differentially expressed genes ( d ) related to fatty acid oxidation and PPAR signaling derived from the bulk RNAseq data of the intestinal organoids from MG53-TG and WT mice. n = 4 for each group. Representative images ( e ) and statistic results of signal intensity of the immunofluorescence staining of MG53 (green) and PPARα (red) determined by Aperio Scanscope scanner and Halo software ( f ), as well as the correlation of the levels of these two proteins ( g ) in the human small intestine of normal subjects ( n = 5) and patients with intestinal inflammation ( n = 8). Nuclei were stained with DAPI (blue); scale bars as indicated. Representative images ( h ) and statistic results of signal intensity of the immunofluorescence staining of MG53 (green) and PPARα (red) determined by Aperio Scanscope scanner and Halo software ( i ), as well as the correlation of the levels of these two proteins ( j ) in the human colon of normal subjects ( n = 4) and patients with intestinal inflammation ( n = 12). Normal distribution was confirmed by Shapiro–Wilk test. Data were analyzed using Mann–Whitney U test ( f , i ) and Pearson correlation analysis ( g , j ). Data were presented as mean ± s.e.m. * p < 0.05 as compared with the corresponding controls Full size image
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Activation of PPARα signaling is required for MG53-induced alleviation of intestinal injury. Body weight change and DAI ( a ) as well as the representative images and statistic results of the intestinal length ( b ) of ISC-MG53-TG and the control MG53 tg-fl mice. n = 7 for each group. Body weight change and DAI ( c ) as well as the representative images and statistic results of the intestinal length ( d ) of the Lgr5 mice with ISC-specific inhibition of MG53 expression via infection with adeno-associated virus with inducible expression of shRNA targeting MG53 (AAV-shMG53) or the control mice infected with control shRNA (AAV-shCON). n = 6 for each group. Body weight change and DAI ( e ) as well as the intestinal length ( f ) of ISC-Ppara-KO mice with or without ISC-specific MG53 overexpression via injecting AAV-MG53 or AAV-CON. n = 8 for each group. Body weight change and DAI ( g ) as well as the intestinal length ( h ) of MG53-KO and control WT mice treated with PPARα agonist fenofibrate or vehicle following DSS treatment. n = 8 for each group. Normal distribution was confirmed by Shapiro–Wilk test. Data were analyzed using two-tailed paired t test ( a – e , g ), Mann–Whitney U test ( f , h ), and were presented as mean ± s.e.m. ns not significant, * p < 0.05 and ** p < 0.01 as compared with the corresponding controls Full size image
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Enhanced PPARα activation by palmitoleic acid contributes to MG53-mediated amelioration of intestinal injury. a Heatmap of free fatty acids (FFA) in MG53-TG and WT intestinal tissues at the indicated time points during DSS treatment. n = 3 for each group. b Changes in the intestinal content of FFA in the MG53-TG and their WT littermates at day 7 after DSS treatment. n = 3 for each group. c Relative change in POA concentrations in the serum of IBD patients in remission ( n = 11) or flare ( n = 26). d Luciferase reporter activity driven by the PPARα-responsive element in the presence of different FFA in HEK293 cells. n = 3 for each group. Body weight change and DAI ( e ) as well as the colon length ( f ) of MG53-TG (TG) and their WT littermates challenged with DSS with or without POA treatment. n = 12 for each group. g Immunofluorescence staining of Ki-67 (red) and statistic results of the length of TA zone of MG53-TG and their WT littermates with or without POA treatment. n = 15 for each group. The nuclei were indicated by DAPI staining (blue); scale bar, 100 μm. h Representative images and statistic results of PAS staining of goblet cells in the colon after POA treatment. n = 10 for each group. Scale bar, 100 μm. Normal distribution was confirmed by Shapiro–Wilk test. Data were analyzed using one-way ANOVA with Tukey post hoc test ( c , e ), two tailed t test ( d , f – h ) and were presented as mean ± s.e.m. ns not significant, * p < 0.05, ** p < 0.01, and *** p < 0.001 as compared with the corresponding controls Full size image
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Immunohistochemical analysis of paraffin-embedded mouse kidney, using Lysozyme Antibody.

Immunohistochemical analysis of paraffin-embedded Rat cerebral cortex, using the Antibody at 1:2000 dilution.

Immunohistochemical analysis of paraffin-embedded Human thyroid cancer, using the Antibody at 1:4000 dilution.

Immunohistochemical analysis of paraffin-embedded Human placenta, using the Antibody at 1:4000 dilution.

Immunohistochemical analysis of paraffin-embedded Mouse skin, using the Antibody at 1:2000 dilution.

Immunohistochemical analysis of paraffin-embedded Mouse heart, using the Antibody at 1:2000 dilution.

Immunofluorescent analysis using the Antibody at 1:50 dilution.