itm2b  (Atlas Antibodies)

Journal: EMBO Molecular Medicine

Article Title: Extracellular vesicle-based targeted protein degradation platform for multiple extracellular proteins

doi: 10.1038/s44321-025-00371-8

Figure Lengend Snippet: ( A ) The uptake levels of specific antibody-blocking EVs (IgG, CD71, SQSTM1, TMEM59, CCN1, ITM2B, ITM2C) were detected after incubation for 12 h. IgG control was used to normalize the grayscale values of each group. Data were analyzed by one-way ANOVA ( n = 3). ( B ) The uptake levels of specific gene-knockdown EVs (Vector, CD71, SQSTM1, TMEM59, CCN1, ITM2B, ITM2C) were detected after incubation for 12 h. Vector control was used to normalize the grayscale values of each group. Data were analyzed by one-way ANOVA ( n = 3). ( C ) Control EVs and SQSTM1 KD EVs were incubated with NPCs for 12 h. Uptake level was analyzed at this time while degradation level was detected following wash out and another 12 h incubation ( n = 3). ( D ) IEM assay to detect the expression of SQSTM1 on HEK-293T derived EVs (scale bar: 100 nm). ( E ) Anti-SQSTM1 antibody was used to pull down HEK-293T derived EVs. IgG was used as negative control. SQSTM1 as well as EV marker-CD63 were detected by western blot. ( F ) Control or SQSTM1 KD EVs were added to NPCs and co-incubated for 12 h. Then, free EVs were wash out and replaced by free mediun containing 50 nM BafA1 for 12 h. After incubation, lysosome enrichment method was used to detect the sorting efficiency of control and SQSTM1 knockdown EVs into lysosomes in NPCs ( n = 3). ( G ) Control or SQSTM1 KD EVs were added to NPCs and co-incubated for 12 h. Then, free EVs were washed out and replaced by free mediun containing 50 nM BafA1 for 12 h. After incubation, immunofluorescence assay was used to detect the co-localization of EGFP with LampII (scale bar: 10 µm) ( n = 6); ( H ) Control or SQSTM1 KD EVs were added to HeLa cells and co-incubated for 12 h. Then, free EVs were washed out and replaced by free mediun containing 50 nM BafA1 for 12 h. After incubation, lysosome enrichment method was used to detect the sorting efficiency of control and SQSTM1 knockdown EVs into lysosomes in HeLa cells ( n = 3); ( I , J ) Analysis of degradation rate ( I ) and lysosomal sorting efficiency ( J ) of control and SQSTM1 overexpression EVs in NPCs ( n = 3); ( K , L ) Analysis of degradation rate ( K ) and lysosomal sorting efficiency (l) of control and SQSTM1 overexpression EVs in HeLa cells ( n = 3). Data were analyzed by one-way ANOVA ( A , B ) unpaired two-tailed t tests ( C , F , G , H , I , J , K , L ). Data were shown as mean ± SD. Each n in ( A , B , C , F , G , H , I , J , K , L ) is biological independent samples. The P values are labeled in the figure.

Article Snippet: ITM2B Polyclonal antibody , Signalway Antibody , 47141.

Techniques: Blocking Assay, Incubation, Control, Knockdown, Plasmid Preparation, Expressing, Derivative Assay, Negative Control, Marker, Western Blot, Immunofluorescence, Over Expression, Two Tailed Test, Labeling

Journal: Neuropathology : official journal of the Japanese Society of Neuropathology

Article Title: Assessing Co-Localization of ITM2B With Alzheimer's Disease and Limbic-Predominant Age-Related TDP-43 Encephalopathy Neuropathologic Changes.

doi: 10.1111/neup.70003

Figure Lengend Snippet: FIGURE 1 | Multiplexed staining of various disease pathologies. A sequential multiplexed staining and analysis, known as QUIVER (Ref [34]), was employed on human FFPE tissue. The procedure started with the staining for IBA1 to assist in image deconvolution and merging. This step uti- lized a permanent chromogen to preserve the staining throughout each subsequent round. Subsequent staining rounds were performed for ITM2B (A and B), AT8 (C), and pTDP-43 (D), sequentially, using a removable chromogen. Post-deconvolution of single-channel IHC images, merged pseudo- fluorescent images were generated for each channel (i). Using HALO software (Indica Labs, version 3.6), a digital markup for each stain was also created (ii) to selectively detect each pathology. To focus on neuronal ITM2B structures (A), the algorithm was turned to omit large plaque-like structures over 1000 μm2 (Bii). Photos captured at 20× magnification. Image deconvolution and markup were completed in HALO software. Arrows denote pathological neuronal ITM2B staining.

Article Snippet: The staining sequence for ensuing rounds included ITM2B (1:100 Atlas Antibodies Cat# TABLE 1 | Demographics of included cases from the University of Kentucky Alzheimer‘s disease research center (UK- ADRC) cohorts.

Techniques: Staining, Generated, Software

Journal: Neuropathology : official journal of the Japanese Society of Neuropathology

Article Title: Assessing Co-Localization of ITM2B With Alzheimer's Disease and Limbic-Predominant Age-Related TDP-43 Encephalopathy Neuropathologic Changes.

doi: 10.1111/neup.70003

Figure Lengend Snippet: FIGURE 2 | Multiplexed staining of ITM2B co-localization in the human hippocampus. A method of sequential multiplexed staining and anal- ysis, known as QUIVER [34], was employed on 16 sections of human FFPE tissue. The procedure began with the staining for IBA1 followed by pT- DP-43 and AT8. ITM2B staining could be observed throughout the entirety of cells, including the apical dendrite co-localized with AT8 in ADNC cases (A). Co-localization was also seen frequently in pure LATE-NC (B). Additionally, cells positive for ITM2B, pTDP-43, and AT8 were seen in ADNC+LATE-NC cases (C). ITM2B-positive neurons that were also double-positive for AT8 and pTDP-43 showed several phenotypes. Their pres- ence was observed in high-intensity ITM2B stained neurons (C) in addition to low-intensity neurons more consistent with the staining pattern ob- served in other regions of the brain (D). Pseudofluorescent images were produced using the object co-localization algorithm in the HALO software. Scale bars = 50 μm. Image insets depict staining before deconvolution.

Article Snippet: The staining sequence for ensuing rounds included ITM2B (1:100 Atlas Antibodies Cat# TABLE 1 | Demographics of included cases from the University of Kentucky Alzheimer‘s disease research center (UK- ADRC) cohorts.

Techniques: Staining, Produced, Software

Journal: Neuropathology : official journal of the Japanese Society of Neuropathology

Article Title: Assessing Co-Localization of ITM2B With Alzheimer's Disease and Limbic-Predominant Age-Related TDP-43 Encephalopathy Neuropathologic Changes.

doi: 10.1111/neup.70003

Figure Lengend Snippet: FIGURE 3 | ITM2B immunostaining. Stained hippocampal sec- tions showed several ITM2B phenotypes in various disease states. Physiological ITM2B staining (low-pathology cases) demonstrated ro- bust immunoreactivity throughout the entire cell in nearly all regions of the hippocampus (A). However, pathological ITM2B could also be observed throughout the hippocampus. In ADNC cases, ITM2B with- in cells could show decreased cytoplasmic reactivity and pronounced puncta throughout the cell (B). Similarly, ITM2B also localized with plaque-like structures resembling compact dense plaques (C) or larg- er, more diffuse plaques (D). Photomicrographs captured at 20× mag- nification. Arrows indicate intraneuronal ITM2B immunoreactive structures. Arrowheads denote ITM2B immunoreactive plaque-like structures.

Article Snippet: The staining sequence for ensuing rounds included ITM2B (1:100 Atlas Antibodies Cat# TABLE 1 | Demographics of included cases from the University of Kentucky Alzheimer‘s disease research center (UK- ADRC) cohorts.

Techniques: Immunostaining, Staining

Journal: Neuropathology : official journal of the Japanese Society of Neuropathology

Article Title: Assessing Co-Localization of ITM2B With Alzheimer's Disease and Limbic-Predominant Age-Related TDP-43 Encephalopathy Neuropathologic Changes.

doi: 10.1111/neup.70003

Figure Lengend Snippet: FIGURE 4 | Digital analysis of ITM2B immunolabeling, stratifying by disease pathology. In addition to ITM2B, levels of AT8+ pTau (A) and pT- DP-43 (B) were quantified in patients with ADNC, LATE-NC, ADNC+LATE-NC, as well as normal control cases. Using the object co-localization algorithm in HALO software, we then quantified the total number of pTDP-43 inclusions that were also positive for AT8 (C).

Article Snippet: The staining sequence for ensuing rounds included ITM2B (1:100 Atlas Antibodies Cat# TABLE 1 | Demographics of included cases from the University of Kentucky Alzheimer‘s disease research center (UK- ADRC) cohorts.

Techniques: Immunolabeling, Control, Software

Journal: Neuropathology : official journal of the Japanese Society of Neuropathology

Article Title: Assessing Co-Localization of ITM2B With Alzheimer's Disease and Limbic-Predominant Age-Related TDP-43 Encephalopathy Neuropathologic Changes.

doi: 10.1111/neup.70003

Figure Lengend Snippet: FIGURE 5 | Digital analysis of ITM2B, pTau, and pTDP-43 colabeling across a spectrum of pathologies. (A): Bar graph showing the density of ITM2B-positive cells per mm2 in different hippocampal subregions: Dentate gyrus (DG), CA3, CA2, CA1, and subiculum (Sub). Data are present- ed for control, ADNC (Alzheimer's disease neuropathologic changes), LATE-NC (Limbic-predominant age-related TDP-43 encephalopathy neuro- pathologic changes), AD+LATE-NC (co-occurrence of both ADNC and LATE-NC). (B): Percentage of ITM2B-positive cells co-localizing with AT8 (a marker for phosphorylated tau, indicating tauopathy). The inset (i) shows a correlation analysis between ITM2B and AT8 markers across all cases, with the linear regression line indicating a positive correlation. (C): Percentage of ITM2B-positive cells co-localizing with pTDP-43 (a marker for phosphorylated TDP-43, associated with LATE-NC). The inset (ii) shows a correlation analysis between ITM2B and pTDP-43 markers across all cas- es, with the linear regression line indicating a trend toward positive correlation.

Article Snippet: The staining sequence for ensuing rounds included ITM2B (1:100 Atlas Antibodies Cat# TABLE 1 | Demographics of included cases from the University of Kentucky Alzheimer‘s disease research center (UK- ADRC) cohorts.

Techniques: Control, Marker

Journal: Neuropathology : official journal of the Japanese Society of Neuropathology

Article Title: Assessing Co-Localization of ITM2B With Alzheimer's Disease and Limbic-Predominant Age-Related TDP-43 Encephalopathy Neuropathologic Changes.

doi: 10.1111/neup.70003

Figure Lengend Snippet: FIGURE 7 | ITM2B co-localization across a range of ADNC severity. Using sequential staining methods for ITM2B and Thioflavin-S, we ob- served several variations of ITM2B reactivity and association with AD pathology. Physiological intraneuronal ITM2B appeared to rarely co-localize with Thio-s (A), however, we also observed heavily punctated forms of ITM2B+ neuronal structures that co-localized with Thio-S+ fibrils, possibly representing a transition stage in the death of the cells (B). We also observed mature neurofibrillary tangles that were not co-localized with ITM2B (C). It is, therefore, possible that as Thio-S levels increase, the levels of ITM2B within a cell decrease, and mature tangles without ITM2B represent a neuron's end stage. Similarly, plaque-like structures could be observed in 3 general stages in diseased brains, including ITM2B+ without Thio-S (D), ITM2B+ with Thio-S (E) or Thio-S+ but ITM2B- (F).

Article Snippet: The staining sequence for ensuing rounds included ITM2B (1:100 Atlas Antibodies Cat# TABLE 1 | Demographics of included cases from the University of Kentucky Alzheimer‘s disease research center (UK- ADRC) cohorts.

Techniques: Staining

Journal: Neuropathology : official journal of the Japanese Society of Neuropathology

Article Title: Assessing Co-Localization of ITM2B With Alzheimer's Disease and Limbic-Predominant Age-Related TDP-43 Encephalopathy Neuropathologic Changes.

doi: 10.1111/neup.70003

Figure Lengend Snippet: FIGURE 6 | Digital Pathological Markup showing ITM2B co-localization in disease-representative cases. After staining for ITM2B, pTDP-43 and AT8 (pTau), a digital markup representing each disease type was generated in HALO software to show the number of ITM2B+ cells also immunore- active for other markers. While ITM2B appeared to co-localize frequently with AT8 staining, it rarely colocalized with pTDP-43. In ADNC+LATE- NC brains, ITM2B occasionally co-localized with cells immunoreactive for both pTDP-43 and AT8. Each red dot represents a single pathological marker. Each black dot is a detected nuclei stained with hematoxylin.

Article Snippet: The staining sequence for ensuing rounds included ITM2B (1:100 Atlas Antibodies Cat# TABLE 1 | Demographics of included cases from the University of Kentucky Alzheimer‘s disease research center (UK- ADRC) cohorts.

Techniques: Staining, Generated, Software, Marker

Journal: Neuropathology : official journal of the Japanese Society of Neuropathology

Article Title: Assessing Co-Localization of ITM2B With Alzheimer's Disease and Limbic-Predominant Age-Related TDP-43 Encephalopathy Neuropathologic Changes.

doi: 10.1111/neup.70003

Figure Lengend Snippet: FIGURE 8 | Western blot analysis of ITM2B and PHF-1 across various subcellular fractions in samples from a normal control and an Alzheimer's disease case. The fractions analyzed include Low Salt (LS), Triton-X treated (TX), Sarcosyl (SARC), and detergent-insoluble, urea-soluble (Urea) fractions. Molecular weight markers are shown on the left. ITM2B (~40kDa expected MW) signals were enriched in the TX and SARC fractions, indicating membrane association, with in- creased PHF-1/pTau but not ITM2B levels in the Urea fraction of the Alzheimer's disease sample. β-Actin is used as a loading control.

Article Snippet: The staining sequence for ensuing rounds included ITM2B (1:100 Atlas Antibodies Cat# TABLE 1 | Demographics of included cases from the University of Kentucky Alzheimer‘s disease research center (UK- ADRC) cohorts.

Techniques: Western Blot, Control, Molecular Weight, Membrane

Journal: Acta Neuropathologica

Article Title: Microglia contribute to the production of the amyloidogenic ABri peptide in familial British dementia

doi: 10.1007/s00401-024-02820-z

Figure Lengend Snippet: Antibodies used in this study

Article Snippet: ITM2B/BRI2 NTF , mouse , Santa cruz sc-374362 , AB_10988049.

Techniques:

Journal: Progress in neurobiology

Article Title: Signal peptide peptidase-like 2b modulates the amyloidogenic pathway and exhibits an Aβ-dependent expression in Alzheimer's disease.

doi: 10.1016/j.pneurobio.2024.102585

Figure Lengend Snippet: Fig. 1. SPPL2b is up-regulated in SH-SY5Y APPswe cells and Aβ42 affects SPPL2b expression. (A) Western blot and immunofluorescence analysis of SPPL2b (Invitrogen, PA5-42683) expression in SH-SY5Y WT (SH WT) and SH-SY5Y APPswe (SH APPswe) cells (SH WT/ SH APPswe, n = 3/3). (B) Quantification of the SPPL2b/Tubulin ratio from the Western blot analysis in A. (C) Western blot analysis of BRI2 (goat Anti-Bri2 BRICHOS antibody) expression in SH-SY5Y WT and APPswe cells. (D) Quantification of mature BRI2 protein (mBRI2, 50 kDa) expression normalized to tubulin protein expression (SH WT/ SH APPswe, n = 3/3). (E) Quantification of the BRI2 cleavage fragment (Frag-BRI2, 35 kDa), (F) and quantification of the BRI2 50 kDa/35 kDa ratio from the Western blot analysis in C (SH WT/ SH APPswe, n = 3/3). (G, H) Analysis of the conditioned media from SH-SY5Y WT and SH-SY5Y APPswe cells using an anti-Bri2 BRICHOS antibody to identifythe soluble BRI2 fragment (sFrag-BRI2) (SH WT/ SH APPswe, n = 5/5). Data from A to H were analyzed by unpaired Student’s t-test. *P<0,05; **P<0,01; ***P<0001; ****P<0,0001 significantly different from SH-SYY WT. (I) Representative Western blot of SPPL2b (Invitrogen, PA5-42683) expression in SH-SY5Y WT cells without (Control) and after 6 hours of exposure to Aβ42 (50 nM, 100 nM). (J) Quantification of SPPL2b expression levels from Western blot analysis in I (Control/50 nM/100 nM, n = 10/4/4). Results are normalized to actin protein expression and analyzed by using one-way ANOVA F (2, 15) = 12,60, followed by Tukey’s multiple comparisons test. *P < 0.05, ***P < 0.001. (K) Representative Western blot of SPPL2b expression in SH-SY5Y APPswe cells without Aβ42 treatment (Control) and after 6 hours of exposure to 10 nM, 50 nM, and 100 nM of Aβ42. (L) Quantification of SPPL2b expression levels from Western blot analysis in K (Control/10 nM/ 50 nM/100 nM, n = 3/3/2/3). Results are normalized to actin protein expression and analyzed by one-way ANOVA F (3, 7) = 10,67, followed by Tukey’s multiple comparisons test. *P < 0.05, **P < 0.01. (M, N) Representative Western blot and analysis of SPPL2b (rabbit anti-SPPL2b) expression in mouse brain cortex kept ex vivo in artificial CSF and treated with Aβ42 50 nM for 6 hours (Control/ 50 nM, n = 6/6). Results are normalized to tubulin protein expression and analyzed by unpaired Student’s t-test. ****P < 0.0001. All data are represented as mean ± S.E.M.

Article Snippet: The arrows indicate the position of the soluble BRI2 (sFrag-BRI2) band location. (F) sFrag-BRI2 relative intensity quantification and (G) the antibodies’ binding regions in the BRI2 protein (biorender.com) (HEK WT/ HEK SPPL2b, n = 3/3). (H) Western blot analysis of cellular APP protein levels in lysates from HEK WT and HEK SPPL2b cells; the mature (mAPP) and soluble (sAPP) forms of APP were detected with the 22C11 antibody. (I, and J) Quantitative analysis of the mAPP and sAPP band intensity normalized to actin (HEK WT/ HEK SPPL2b, n = 3/3). (K) Quantification of the ratio between mAPP and sAPP levels determined by the Western blot analysis in (E). (L) Representative Western blot of sAPP levels in conditioned media from HEK WT cells and HEK cells overexpressing SPPL2b and (M) quantifying band intensities (HEK WT/ HEK SPPL2b, n = 3/3). (N) Immunofluorescence staining of BRI2 (Anti-ITM2B Antibody (C-8), Santa Cruz) in cultured mouse primary neurons derived from WT and SPPL2b KO embryos and (O) quantification of BRI2 intensity (WT/ SPPL2b KO, n = 5/4).

Techniques: Expressing, Western Blot, Immunofluorescence, Control, Ex Vivo

Journal: Progress in neurobiology

Article Title: Signal peptide peptidase-like 2b modulates the amyloidogenic pathway and exhibits an Aβ-dependent expression in Alzheimer's disease.

doi: 10.1016/j.pneurobio.2024.102585

Figure Lengend Snippet: Fig. 5. SPPL2b is mainly expressed in neurons and microglia associated with Aβ plaques. (A) In the left image is a representative immunofluorescence staining of SPPL2b (green) and Aβ plaques by using the anti-Aβ antibody 82E1 (red) in 10 months old AppNL-G-F mice. In the second left image is a representative SPPL2b staining in vicinity of an Aβ plaque shown. On the right, third and fourth images, immunohistochemistry staining of SPPL2b (in red) and Aβ plaques by using the 6E10 antibody (in blue) in 10 months old AppNL-G-F mice. (B) SPPL2b immunofluorescence staining (green) and NeuN staining (red) to visualize neurons and Hoechst for nuclear staining (blue), (C) SPPL2b (green) and Iba1 (red) immunofluorescence staining together with FSB to stain Aβ plaques (D) SPPL2b (green) and GFAP (red) immunofluorescence staining together with FSB to stain Aβ plaques. Scale bar sizes are denoted in the figure.

Article Snippet: The arrows indicate the position of the soluble BRI2 (sFrag-BRI2) band location. (F) sFrag-BRI2 relative intensity quantification and (G) the antibodies’ binding regions in the BRI2 protein (biorender.com) (HEK WT/ HEK SPPL2b, n = 3/3). (H) Western blot analysis of cellular APP protein levels in lysates from HEK WT and HEK SPPL2b cells; the mature (mAPP) and soluble (sAPP) forms of APP were detected with the 22C11 antibody. (I, and J) Quantitative analysis of the mAPP and sAPP band intensity normalized to actin (HEK WT/ HEK SPPL2b, n = 3/3). (K) Quantification of the ratio between mAPP and sAPP levels determined by the Western blot analysis in (E). (L) Representative Western blot of sAPP levels in conditioned media from HEK WT cells and HEK cells overexpressing SPPL2b and (M) quantifying band intensities (HEK WT/ HEK SPPL2b, n = 3/3). (N) Immunofluorescence staining of BRI2 (Anti-ITM2B Antibody (C-8), Santa Cruz) in cultured mouse primary neurons derived from WT and SPPL2b KO embryos and (O) quantification of BRI2 intensity (WT/ SPPL2b KO, n = 5/4).

Techniques: Immunofluorescence, Staining, Immunohistochemistry