mouse anti catd  (Santa Cruz Biotechnology)

Journal: The Journal of Neuroscience

Article Title: Loss of Christianson Syndrome Na + /H + Exchanger 6 (NHE6) Causes Abnormal Endosome Maturation and Trafficking Underlying Lysosome Dysfunction in Neurons

doi: 10.1523/JNEUROSCI.1244-20.2021

Figure Lengend Snippet: Loss of NHE6 increases MVB fusion with the PM and exosome secretion. A , Representative TIRF image depicting MVB-PM fusion and exosome release as developed from . Widefield image of neuron cotransfected with mCherry and CD63-pHluorin expression constructs. White inset, Location of MVB-PM fusion and the zoomed in panels on the right. Each panel represents the progression of a CD63-pHluorin fusion event with the PM with the number of seconds indicated below the panel. Scale bars: large, 10 µm; small, 1 µm. B , Quantification of full MVB-PM fusion/exosome release events per cell over 5 min in WT and Nhe6 -/Y male littermate mouse primary hippocampal neurons at 14 DIV (WT n = 28 cells from 7 mice, Nhe6 -/Y n = 18 cells from 7 mice, 5 litters, p = 0.009, Glass's Δ = 2.27). C , Quantification of full MVB-PM fusion/exosome release events per cell over 5 min in WT and Nhe6 -/Y male mouse primary hippocampal neurons at 14 DIV under the following conditions: untreated (same as in B ), U18666A (positive control) (WT n = 14 cells from 5 mice, Nhe6 -/Y n = 14 cells from 5 mice, 3 litters), bafilomycin A1 (positive control) (WT n = 14 cells from 7 mice, Nhe6 -/Y n = 16 cells from 6 mice, 4 litters, Kruskal–Wallis test with Dunn's test: WT untreated compared with WT bafilomycin A1 p = 0.002, Glass's Δ = 3.04). D , E , CD63 western blot ( D ) and quantification ( E ) in WT and Nhe6 -/Y male littermate mouse primary hippocampal neurons at 14 DIV (WT n = 5 cultures, Nhe6 -/Y n = 5 cultures, 5 litters, p = 0.02, Glass's Δ = 1.68). F , Released β-Hex enzyme activity following short-term incubation in Tyrode's solution followed by treatment with either ionomycin or DMSO (WT n = 9, Nhe6 -/Y n = 9, 8 litters). G , Released CatD enzyme activity following short-term incubation in Tyrode's solution followed by treatment with either ionomycin or DMSO (WT n = 6, Nhe6 -/Y n = 6, 5 litters). H , Released LDH activity across all β-Hex (WT n = 5, Nhe6 -/Y n = 5, 5 litters) and CatD (WT n = 4, Nhe6 -/Y n = 4, 3 litters) experiments. Data are mean ± SEM. Unpaired two-tailed Student's t test ( C , WT- Nhe6 -/Y : bafilomycin A1) with Welch's correction ( E ), Mann–Whitney test ( B , C , WT- Nhe6 -/Y : U18666A), Kruskal–Wallis test with Dunn's test ( C , differences between treatments by genotype), two-way ANOVA with Tukey's multiple comparisons test ( F , G , H ).

Article Snippet: The following antibodies were used for western blot: actin (Sigma, A3853, Ms, 1:1000), CatD (R&D Systems, AF1029, Gt, 1:1000), CD63 (Abcam, EPR21151-ab217345, Rb, 1:1000), ci-mannose 6-phosphate receptor (M6PR) (Cell Signaling Technology, 14364S, Rb, 1:500), GAPDH (Sigma, G8795, Ms, 1:40 000), LAMP1 (DSHB, 1D4B, Rt, 1:1000), RAB5 (Cell Signaling Technology, 3547, Rb, 1:1000), and RAB7 (Sigma, R8779, Ms, 1:1000).

Techniques: Expressing, Construct, Positive Control, Western Blot, Activity Assay, Incubation, Two Tailed Test, MANN-WHITNEY

Journal: The EMBO Journal

Article Title: Loss of TREM2 rescues hyperactivation of microglia, but not lysosomal deficits and neurotoxicity in models of progranulin deficiency

doi: 10.15252/embj.2021109108

Figure Lengend Snippet: Expression of all analyzed gene transcripts in GRN −/− hiMGL treated with control isotype antibody, Ab1, or Ab2 in comparison to WT hiMGL. Data show the mean of four individual treatments and NanoString measurements. The mRNA counts for each gene were normalized to the mean value of all WT samples followed by a log2 transformation. Volcano plot presentation of the differently expressed transcripts in GRN −/− hiMGL treated with isotype compared to WT hiMGL. Genes with more than 20% significantly changed expression are marked in purple (upregulated) or blue (downregulated). Volcano plot presentation of the differently expressed transcripts in GRN −/− hiMGL treated with Ab1 comparison to WT hiMGL. Genes with more than 20% significantly changed expression are marked in purple (upregulated) or blue (downregulated). Volcano plot presentation of the differently expressed transcripts in GRN −/− hiMGL treated with Ab2 comparison to WT hiMGL. Genes with more than 20% significantly changed expression are marked in purple (upregulated) or blue (downregulated). Transcript levels of DAM gene transcripts significantly altered in GRN −/− hiMGL, treated with Ab1 or Ab2 in comparison to isotype treatment from the data set in A, and normalized to the mean of the WT hiMGL samples ( n = 4, biological replicates). Transcript levels of CTSD , NPC2 , and CD68 of WT and GRN −/− hiMGL untreated, treated with isotype control, and Ab1 or Ab2 in comparison to WT hiMGL from the data set in A normalized to the mean of the WT hiMGL samples ( n = 4, biological replicates). Catalytic activity of cathepsin D (CatD) in untreated WT and GRN −/− hiMGL or GRN −/− hiMGL treated with isotype control, Ab1 or Ab2 (20 μg/ml, 40 μg/ml), as measured by a CatD in vitro activity assay ( n = 3, biological replicates). Data information: Data represent mean ± SEM. For statistical analysis in B–D, the unpaired, two‐tailed student’s t ‐test was performed, in E, one‐way ANOVA with Dunnett’s post hoc test was used to compare Ab1 and Ab2 (20 µg/ml and 40 µg/ml) conditions to the isotype‐treated condition, and in F and G, one‐way ANOVA with Dunnett`s post hoc test was used to compare Ab1‐, Ab2‐ (20 µg/ml and 40 µg/ml), and isotype‐treated condition to WT cells. Statistical significance was set at * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001, ***** P < 0.00001, and ns, not significant. Source data are available online for this figure.

Article Snippet: Proteins of interest were detected using the following primary antibodies: goat anti‐TREM2 (R&D Systems, Inc., #AF1828), rabbit anti‐PGRN (ThermoFisher, #40‐3400), goat anti‐CatD (R&D, #AF1029), mouse anti‐SPH (abcam, # ab8049), mouse anti‐βActin (Sigma, #A5316), mouse anti‐GAPDH (Invitrogen, #AM4300), and rabbit anti‐Calnexin (Stressgene, #SPA‐860) followed by incubation with horseradish peroxidase‐conjugated secondary antibodies and ECL Plus substrate (ThermoFisher, Pierce ECL Plus Western Blotting Substrates).

Techniques: Expressing, Transformation Assay, Activity Assay, In Vitro, Two Tailed Test

Journal: The EMBO Journal

Article Title: Loss of TREM2 rescues hyperactivation of microglia, but not lysosomal deficits and neurotoxicity in models of progranulin deficiency

doi: 10.15252/embj.2021109108

Figure Lengend Snippet: A Western blot of CatD in total brain lysates from 14‐month‐old female WT, Grn −/− , Trem2 −/− , and Double −/− mice. CatD maturation variants are indicated (sc: single chain; hc: heavy chain; n = 3). B, C Quantification of CatD variants in A normalized to WT ( n = 3 per genotype). D, E Catalytic activity of CatD in brain lysates from female 6‐month‐old ( n = 4 per genotype) (D) or 14‐month‐old ( n = 3 per genotype) (E) Grn −/− , Trem2 −/− , and Double −/− mice normalized to WT. F Immunohistochemical analysis of lipofuscin (green) in coronal brain sections. Representative images of thalamus are shown. Scalebars = 50 μm. G Quantification of lipofuscin autofluorescence. Five images per mouse were taken, and means were normalized to WT samples ( n = 3 per genotype, female). Data information: Data represent mean ± SEM. For statistical analysis, one‐way ANOVA with Tukey’s post hoc test of Grn −/− , Trem2 −/− , and Double −/− was used. Statistical significance was set at * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001, and ns, not significant. Source data are available online for this figure.

Article Snippet: Proteins of interest were detected using the following primary antibodies: goat anti‐TREM2 (R&D Systems, Inc., #AF1828), rabbit anti‐PGRN (ThermoFisher, #40‐3400), goat anti‐CatD (R&D, #AF1029), mouse anti‐SPH (abcam, # ab8049), mouse anti‐βActin (Sigma, #A5316), mouse anti‐GAPDH (Invitrogen, #AM4300), and rabbit anti‐Calnexin (Stressgene, #SPA‐860) followed by incubation with horseradish peroxidase‐conjugated secondary antibodies and ECL Plus substrate (ThermoFisher, Pierce ECL Plus Western Blotting Substrates).

Techniques: Western Blot, Activity Assay, Immunohistochemical staining

Journal: bioRxiv

Article Title: Loss of TREM2 reduces hyperactivation of progranulin deficient microglia but not lysosomal pathology

doi: 10.1101/2021.07.08.451574

Figure Lengend Snippet: A. Expression of all analyzed gene transcripts in GRN -/- hiMGL treated with control isotype antibody, Ab1 or Ab2 in comparison to WT hiMGL. Data show the mean of four individual NanoString measurements. The mRNA counts for each gene were normalized to the mean value of all WT samples followed by a log2 transformation. B. Volcano blot presentation of the differently expressed transcripts in GRN -/- hiMGL treated with isotype compared to WT hiMGL. Genes with more than 20% significantly changed expression are marked in purple (upregulated) or blue (downregulated). C. Volcano blot presentation of the differently expressed transcripts in GRN -/- hiMGL treated with Ab1 comparison to WT hiMGL. Genes with more than 20% significantly changed expression are marked in purple (upregulated) or blue (downregulated). D. Volcano blot presentation of the differently expressed transcripts in GRN -/- hiMGL treated with Ab2 comparison to WT hiMGL. Genes with more than 20% significantly changed expression are marked in purple (upregulated) or blue (downregulated). E. Transcript levels of TREM2 in GRN -/- hiMGL treated with isotype control, antagonistic Ab1 and Ab2 normalized to the mean of the WT hiMGL samples. F. Levels of DAM gene transcripts significantly altered in GRN -/- hiMGL treated with Ab1 or Ab2 in comparison to isotype treatment from the data set in A normalized to the mean of the WT hiMGL samples. G. Transcript levels of CTSD and CD68 of GRN -/- hiMGL treated with isotype control, Ab1 or Ab2 in comparison to WT hiMGL from the data set in A normalized to the mean of the WT hiMGL samples. H. Catalytic activity of cathepsin D (CatD) in untreated WT and GRN -/- hiMGL or GRN -/- hiMG treated with isotype control, Ab1 or Ab2 (20 μg/ml, 40 μg/ml), as measured by a CatD in vitro activity assay (n=3). Data represent mean ± SEM. For statistical analysis in B - D the unpaired, two-tailed student’s t-test was performed, in E and F one-way ANOVA with Dunnett’s post hoc test was used to compare Ab1 and Ab2 (20 µg/ml and 40µg/ml) conditions to the isotype treated condition, and in G - H one-way ANOVA with Dunnett’s post hoc test was used to compare Ab1, Ab2 (20 µg/ml and 40µg/ml) and isotype treated condition to WT cells. Statistical significance was set at *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001, and ns, not significant.

Article Snippet: Proteins of interest were detected using the following primary antibodies: goat anti-TREM2 (R&D Systems, Inc., #AF1828), rabbit anti-PGRN (ThermoFisher, #40-3400), goat anti-CatD (R&D, #AF1029), mouse anti-βActin (Sigma, #A5316) and rabbit anti-Calnexin (Stressgene, #SPA-860) followed by incubation with horseradish peroxidase-conjugated secondary antibodies and ECL Plus substrate (ThermoFisher, Pierce ECL Plus Western Blotting Substrates).

Techniques: Expressing, Transformation Assay, Activity Assay, In Vitro, Two Tailed Test

Journal: bioRxiv

Article Title: Loss of TREM2 reduces hyperactivation of progranulin deficient microglia but not lysosomal pathology

doi: 10.1101/2021.07.08.451574

Figure Lengend Snippet: A. Western blot of CatD in total brain lysates from 14-month-old WT, Grn -/- , Trem2 -/- , and Double -/- mice. CatD maturation variants are indicated (sc: single chain; hc: heavy chain; n=3). B., C. Quantification of CatD variants in A normalized to WT. D., E. Catalytic activity of CatD in brain lysates from 6-month-old ( D ) or 14-month-old ( E ) Grn -/- , Trem2 -/- , Double -/- mice normalized to WT (n=3 per genotype). F. Immunohistochemical analysis of lipofuscin (green) in coronal brain sections. Representative images of hypothalamus. Scalebars = 50 μm. G. Quantification of lipofuscin autofluorescence. Five pictures per mouse were taken, means were normalized to WT samples (n=3 per genotype). Data represent mean ± SEM. For statistical analysis one-way ANOVA with Tukey’s post hoc test of Grn -/- , Trem2 -/- and Double -/- was used. Statistical significance was set at *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001, and ns, not significant.

Article Snippet: Proteins of interest were detected using the following primary antibodies: goat anti-TREM2 (R&D Systems, Inc., #AF1828), rabbit anti-PGRN (ThermoFisher, #40-3400), goat anti-CatD (R&D, #AF1029), mouse anti-βActin (Sigma, #A5316) and rabbit anti-Calnexin (Stressgene, #SPA-860) followed by incubation with horseradish peroxidase-conjugated secondary antibodies and ECL Plus substrate (ThermoFisher, Pierce ECL Plus Western Blotting Substrates).

Techniques: Western Blot, Activity Assay, Immunohistochemical staining