mouse progranulin  (R&D Systems)

Journal: bioRxiv

Article Title: Granulin loss and TMEM106B risk converge on lysosomal C-terminal fragment pathology in frontotemporal dementia

doi: 10.64898/2026.03.25.713523

Figure Lengend Snippet: A. Western blots show that recombinant progranulin reduces TMEM106B CTF accumulation in GRN KO iNeurons in a dose-dependent manner. Cells were treated with recombinant progranulin for three days before harvest. B. Quantification of TMEM106B dimers, monomers, and CTFs from panel A. Normalized ratios were calculated by dividing the intensity of each TMEM106B species (dimer, monomer, or CTF) by the loading control (GAPDH), then normalizing to the first bar (0 nM progranulin). C. Western blots show that BSA treatment does not reduce TMEM106B CTF accumulation in GRN KO iNeurons in a dose-dependent manner. Cells were treated with BSA for three days before harvest. D. Quantification of TMEM106B dimers, monomers, and CTFs from panel C. Normalized ratios were calculated by dividing the intensity of each TMEM106B species (dimer, monomer, or CTF) by the loading control (GAPDH), then normalizing to the first bar (0 nM BSA). Bar plots represent the mean, and each dot represents a replicate (n = 3 replicates per condition). Statistical significance was determined by a two-sided Welch’s t-test: ns (not significant), p > 0.05; *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001.

Article Snippet: Primary antibodies used in this work with dilution information are as follows: TMEM106B (E7H7Z) antibody (1:500; Cell Signaling Technology, 93334), cleaved TMEM106B (Ser120) antibody (1:500; Cell Signaling Technology, 87145), C-terminal TMEM106B antibody (1:1000, created in the Dr. Leonard Petrucelli laboratory), GAPDH (1:2000; Sigma-Aldrich, G8795), Histone H3 antibody (1:5000; Abcam, ab1791), Beta-tubulin antibody (1:40000, Sigma-Aldrich, 66240-1-Ig), Human LAMP1 antibody (1:1000, Cell Signaling Technology, 9091P or 15665S), Mouse LAMP1 antibody (1:1000, DSHB, 1D4B), progranulin antibody (1:1000, R&D Systems, AF2420), CTS B (1:1000, Cell Signaling Technology, 31718T), PDI antibody (1:1000, Enzo Life Sciences, ADI-SPA-891-D), Citrate synthase antibody (1:1000, Cell Signaling Technology, 14309T), Golgin-97 antibody (1:1000, Cell Signaling Technology, 13192T), HA-Tag (C29F4) antibody (1:1000, Cell Signaling Technology, 3724S), Catalase antibody (1:1000, Cell Signaling Technology, D4P7B), GFP antibody (1:2000, Antibodies Incorporated, 75-131), and V5 antibody (1:1000, Thermo Fisher Scientific, R960-25).

Techniques: Western Blot, Recombinant, Control

Journal: bioRxiv

Article Title: Granulin loss and TMEM106B risk converge on lysosomal C-terminal fragment pathology in frontotemporal dementia

doi: 10.64898/2026.03.25.713523

Figure Lengend Snippet: A. Left panel: Schematic of TMEM106B showing the T185S coding variant (rs3173615) located in the C-terminal domain. Right panel: Isogenic iPSC-derived neurons were generated with three genotypes: CC (homozygous threonine, TT), CG (heterozygous threonine/serine, TS), and GG (homozygous serine, SS). B. Western blots show that the copy number of the protective S185 allele anti-correlates with TMEM106B CTF levels in the lysosome. Purified lysosomes were from GRN WT iNeurons with TT, TS, or SS genotypes. Lysosomes were purified by immunoprecipitation using the LysoTag. C. Quantification of TMEM106B dimers, monomers, and CTFs from panel B. Normalized ratios were calculated by dividing the intensity of each TMEM106B species (dimer, monomer, or CTF) by the loading control (LAMP1), then normalizing to the first bar (TT genotype) of each TMEM106B species. D. Western blots show that GRN KO increases TMEM106B CTF levels in iNeurons with SS or TT genotypes. Whole cell lysates were analyzed. E. Quantification of TMEM106B CTFs from panel D. Normalized ratios were calculated by dividing the intensity of TMEM106B CTF by the loading control (beta-tubulin) and then normalizing to the first bar. F. Western blots show that recombinant progranulin treatment reduces TMEM106B CTF accumulation in iNeurons with SS, TS, or TT genotypes in a dose-dependent manner. Cells were treated with recombinant progranulin for three days before harvest. G. Quantification of TMEM106B dimers and CTFs from panel F. Normalized ratios were calculated by dividing the intensity of each TMEM106B species (dimer or CTF) by the loading control (beta-tubulin), then normalizing to the first bar (0 nM progranulin) of each genotype group. Bar plots represent the mean, and each dot represents a replicate (n = 3 replicates per condition). Statistical significance was determined by two-sided Welch’s t-test: ns (not significant), p > 0.05; *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001.

Article Snippet: Primary antibodies used in this work with dilution information are as follows: TMEM106B (E7H7Z) antibody (1:500; Cell Signaling Technology, 93334), cleaved TMEM106B (Ser120) antibody (1:500; Cell Signaling Technology, 87145), C-terminal TMEM106B antibody (1:1000, created in the Dr. Leonard Petrucelli laboratory), GAPDH (1:2000; Sigma-Aldrich, G8795), Histone H3 antibody (1:5000; Abcam, ab1791), Beta-tubulin antibody (1:40000, Sigma-Aldrich, 66240-1-Ig), Human LAMP1 antibody (1:1000, Cell Signaling Technology, 9091P or 15665S), Mouse LAMP1 antibody (1:1000, DSHB, 1D4B), progranulin antibody (1:1000, R&D Systems, AF2420), CTS B (1:1000, Cell Signaling Technology, 31718T), PDI antibody (1:1000, Enzo Life Sciences, ADI-SPA-891-D), Citrate synthase antibody (1:1000, Cell Signaling Technology, 14309T), Golgin-97 antibody (1:1000, Cell Signaling Technology, 13192T), HA-Tag (C29F4) antibody (1:1000, Cell Signaling Technology, 3724S), Catalase antibody (1:1000, Cell Signaling Technology, D4P7B), GFP antibody (1:2000, Antibodies Incorporated, 75-131), and V5 antibody (1:1000, Thermo Fisher Scientific, R960-25).

Techniques: Variant Assay, Derivative Assay, Generated, Western Blot, Purification, Immunoprecipitation, Control, Recombinant

Journal: bioRxiv

Article Title: Granulin loss and TMEM106B risk converge on lysosomal C-terminal fragment pathology in frontotemporal dementia

doi: 10.64898/2026.03.25.713523

Figure Lengend Snippet: A. Box plots showing that the common GRN risk variant (rs5848-T) is significantly associated with reduced progranulin protein levels. The association analysis was performed as using limma, adjusting for sex, postmortem interval, and final consensus cognitive diagnosis. B. Box plots showing that the protective TMEM106B rs3173615 allele is significantly associated with reduced levels of peptides mapping to the TMEM106B C-terminal domain (CTD) in the second ROSMAP cohort (ROSMAP-R2). Association analysis was performed using a linear regression model adjusted for sample batch, sex, race, age of death, postmortem interval, and APOE genotype. C. Box plots showing that the protective TMEM106B rs3173615 allele is significantly associated with increased levels of peptides mapping to the TMEM106B N-terminal domain (NTD) in the second ROSMAP cohort (ROSMAP-R2). The association analysis was performed as in panel B. D. Box plots showing that the common GRN risk variant (rs5848-T) is not significantly associated with increased levels of TMEM106B CTD-mapping peptides in the second ROSMAP cohort (ROSMAP-R2). The association analysis was performed as in panel B. E. Box plots showing that the common GRN variant (rs5848-T) is not significantly associated with levels of TMEM106B NTD-mapping peptides in the second ROSMAP cohort (ROSMAP-R2). The association analysis was performed as in panel B. F. Covariate-adjusted box plots illustrating that the protective TMEM106B rs3173615 allele is associated with a stepwise reduction of CTD-mapping peptides across all GRN genotypes in in the first ROSMAP cohort (ROSMAP-R1), displaying the independent, additive effects of both variants. G. Covariate-adjusted box plots showing N-terminal TMEM106B peptides across GRN genotypes in harmonized ROSMAP data. Details of the statistical analysis can be found in the Methods section. Statistical significance was determined by two-sided Welch’s t-test: ns (not significant), p > 0.05; *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001.

Article Snippet: Primary antibodies used in this work with dilution information are as follows: TMEM106B (E7H7Z) antibody (1:500; Cell Signaling Technology, 93334), cleaved TMEM106B (Ser120) antibody (1:500; Cell Signaling Technology, 87145), C-terminal TMEM106B antibody (1:1000, created in the Dr. Leonard Petrucelli laboratory), GAPDH (1:2000; Sigma-Aldrich, G8795), Histone H3 antibody (1:5000; Abcam, ab1791), Beta-tubulin antibody (1:40000, Sigma-Aldrich, 66240-1-Ig), Human LAMP1 antibody (1:1000, Cell Signaling Technology, 9091P or 15665S), Mouse LAMP1 antibody (1:1000, DSHB, 1D4B), progranulin antibody (1:1000, R&D Systems, AF2420), CTS B (1:1000, Cell Signaling Technology, 31718T), PDI antibody (1:1000, Enzo Life Sciences, ADI-SPA-891-D), Citrate synthase antibody (1:1000, Cell Signaling Technology, 14309T), Golgin-97 antibody (1:1000, Cell Signaling Technology, 13192T), HA-Tag (C29F4) antibody (1:1000, Cell Signaling Technology, 3724S), Catalase antibody (1:1000, Cell Signaling Technology, D4P7B), GFP antibody (1:2000, Antibodies Incorporated, 75-131), and V5 antibody (1:1000, Thermo Fisher Scientific, R960-25).

Techniques: Variant Assay, Biomarker Discovery

Journal: Biochemistry and Biophysics Reports

Article Title: Accumulation of prosaposin and progranulin around the subfornical organ induces polydipsia in SAP-D-deficient mice

doi: 10.1016/j.bbrep.2025.102388

Figure Lengend Snippet: Increased protein levels of PSAP and PGRN within the SFO of SAP-D −/− mice a – b) , Coronal brain section at 0.7–0.8 mm posterior to bregma containing the SFO. a: DAPI staining. a-2: Enlarged view of the white square in a-1. SFO: subfornical organ, 3V: third ventricle. b; Double immunofluorescent staining of PSAP (red) and PGRN (green) in 10-month-old female WT and SAP-D −/− mice. DAPI (blue) staining showed the nuclei ( a and b ). Scale bar, 500 μm ( a and b ). White arrowheads indicate the SFO regions ( b ). c) Cerebral region from 3-, 6-, and 10-month-old male and female mice containing the SFO (0.7–0.8 mm posterior to ∗bregma) used for protein extraction and Western blot ( d – h ) using anti- PSAP, PGRN, and GAPDH antibodies. Quantification normalized to GAPDH expression and represented as the mean ± SD of three mice for each group. and indicate the individual values in each group ( e , f , h ). d – f) PSAP and PGRN protein levels in the SFO were remarkably increased. Their quantification by densitometric analysis is represented in e for male and f for female, respectively. e ) For PSAP/GAPDH, two-way ANOVA revealed a significant main effects of genotype (F(1,24) = 547.7, p < 0.0001, ηp 2 = 0.48, 95 % CI [−74.51, −62.43]), with no effect of age ( p = 0.46) or genotype × age interaction ( p = 0.47). Post-hoc Tukey's tests showed that SAP-D −/− differed from WT at 3 M ( p < 0.0001, Cohen's d = 9.99, 95 % CI [−86.30, −54.96]), 6 M ( p < 0.0001, Cohen's d = 7.24, 95 % CI [−79.04, −47.70]), and 10 M ( p < 0.0001, Cohen's d = 8.77, 95 % CI [−87.09, −55.75]). For PGRN/GAPDH, two-way ANOVA revealed a significant main effect of genotype (F(1,24) = 354.2, p < 0.0001, ηp 2 = 0.48, 95 % CI [−46.39, −37.22]), with no effect of age ( p = 0.73) or genotype × age interaction ( p = 0.76). Post-hoc Tukey's tests showed that SAP-D −/− differed from WT at 3 M ( p < 0.0001, Cohen's d = 7.87, 95 % CI [−52.71, −28.91]), 6 M ( p < 0.0001, Cohen's d = 5.35, 95 % CI [−52.82, −29.02]), and 10 M ( p < 0.0001, Cohen's d = 8.77, 95 % CI [−52.32, −28.52]). f ) For PSAP/GAPDH, two-way ANOVA revealed a significant main effect of genotype (F(1,24) = 611.1, p < 0.0001, ηp 2 = 0.48, 95 % CI [−74.42, −62.95]), with no effect of age ( p = 0.07) or genotype × age interaction (p = 0.07). Post-hoc Tukey's tests showed that SAP-D −/− differed from WT at 3 M ( p < 0.0001, Cohen's d = 9.99, 95 % CI [−86.30, −54.96]), 6 M ( p < 0.0001, Cohen's d = 7.24, 95 % CI [−79.04, −47.70]), and 10 M ( p < 0.0001, Cohen's d = 8.77, 95 % CI [−87.09, −55.75]). For PGRN/GAPDH, two-way ANOVA revealed a significant main effect of genotype (F(1,24) = 273.5, p < 0.0001, ηp 2 = 0.47, 95 % CI [−63.96, −49.76]), with no effect of age ( p = 0.71) or genotype × age interaction (p = 0.70). Post-hoc Tukey's tests showed that SAP-D −/− differed from WT at 3 M ( p < 0.0001, Cohen's d = 7.31, 95 % CI [−73.43, −36.60]), 6 M ( p < 0.0001, Cohen's d = 5.71, 95 % CI [−73.04, −36.22]), and 10 M ( p < 0.0001, Cohen's d = 5.35, 95 % CI [−73.08, −36.26]). g – h ) Comparison of PSAP and PGRN protein expression in the SFO, whole cerebrum, and cerebellum. The quantitative analysis is shown in h . h ) For PSAP/GAPDH, one-way ANOVA revealed a significant effect in SAP-D −/− mice (F(2,6) = 30.06, p = 0.0007, η 2 = 0.90), but not in WT mice ( p = 0.3461). Tukey's post hoc tests showed significant differences for SFO versus cerebellum ( p = 0.0010, Cohen's d = 5.26, 95 % CI [46.80, 119.1]) as well as and cerebrum versus cerebellum ( p = 0.0018, Cohen's d = 9.34, 95 % CI [38.39, 110.6]). There was no significant difference for SFO versus cerebrum ( p = 0.76, Cohen's d = 0.47, 95 % CI [−27.71, 44.54]). For PGRN/GAPDH, one-way ANOVA revealed a significant effect in SAP-D −/− mice (F(2,6) = 54.42, p = 0.0001, η 2 = 0.94), but not in WT mice ( p = 0.6327). Tukey's post hoc tests showed significant differences for SFO versus cerebrum ( p = 0.0005, Cohen's d = 5.43, 95 % CI [26.19, 57.84]) as well as and SFO versus cerebellum ( p = 0.0005, Cohen's d = 5.43, 95 % CI [26.19, 57.84]), but not for cerebrum versus cerebellum ( p = 0.3274, Cohen's d = 6.06, 95 % CI [−7.72, 23.92]). ns: no significant difference. ∗∗∗∗ p < 0.0001. ∗∗∗ p < 0.001. ∗∗ p < 0.01.

Article Snippet: Membranes were blocked with 5 % nonfat milk in TBS-T (1 % Tween 20) overnight at 4 °C and incubated in the appropriate primary antibody in 3 % BSA in TBS-T for 2 h at room temperature shaking with PSAP (1:1,000, 10801-1-AP, Proteintech, USA), PGRN (1:1,000, AF 2557, R&D Systems, USA), and GAPDH (1:5000 60004-1-Ig, proteintech, USA).

Techniques: Staining, Protein Extraction, Western Blot, Expressing, Comparison

Journal: Biochemistry and Biophysics Reports

Article Title: Accumulation of prosaposin and progranulin around the subfornical organ induces polydipsia in SAP-D-deficient mice

doi: 10.1016/j.bbrep.2025.102388

Figure Lengend Snippet: Increased PSAP and PGRN immunostaining in the SFO and its surrounding tissues in SAP-D −/− mice a) Double immunofluorescent staining of PSAP (red) and PGRN (green) around the SFO in 10-month-old female WT and SAP-D −/− mice. The white dotted lines enclose the SFO. b ) enlarged white ⅰ-iv squares in a, as indicated. White arrowheads indicate co-staining with anti-PGRN and PSAP antibodies. Open arrowheads indicate staining with PGRN alone. Nuclei are labeled by DAPI (blue) staining. All scale bars, 20 μm.

Article Snippet: Membranes were blocked with 5 % nonfat milk in TBS-T (1 % Tween 20) overnight at 4 °C and incubated in the appropriate primary antibody in 3 % BSA in TBS-T for 2 h at room temperature shaking with PSAP (1:1,000, 10801-1-AP, Proteintech, USA), PGRN (1:1,000, AF 2557, R&D Systems, USA), and GAPDH (1:5000 60004-1-Ig, proteintech, USA).

Techniques: Immunostaining, Staining, Labeling

Journal: Biochemistry and Biophysics Reports

Article Title: Accumulation of prosaposin and progranulin around the subfornical organ induces polydipsia in SAP-D-deficient mice

doi: 10.1016/j.bbrep.2025.102388

Figure Lengend Snippet: Infiltration of CD68-positive activated microglia/macrophages co-expressing PSAP and PGRN into the SFO and surrounding tissues a – b) Double immunofluorescent staining of PGRN (green) and CD68 (red) around the SFO in 10-month-old-female WT and SAP-D −/− mice. b , Magnified images of the indicated white squares in a . ⅰ: SFO, ⅱ: Fornix, and ⅲ: Perivascular, bv: blood vessel. c ) Quantification of PGRN- and/or CD68-staining in the SFO and surrounding areas in WT and SAP-D −/− mice. Data are shown as the mean ± SD (n = 3). The left panel presents a stacked bar chart, whereas the right panel shows the individual data values in a bar chart format. d – h ) Triple immunofluorescent staining of PSAP (red), PGRN (green), and CD68 (cyan) around the SFO in 10-month-old-female SAP-D −/− mice. e) Magnified images of the indicated white squares in d) ⅳ: Boundary, ⅴ: Fornix, and ⅵ: Perivascular. White arrowheads indicate triple co-staining with PSAP, PGRN, and CD68. Open arrowheads indicate PGRN signals alone. Co-localization rates of CD68 positive areas in PSAP ( f ), PGRN ( g ), and PSAP-PGRN staining areas ( h ) around the SFO of SAP-D −/− mice, respectively. f-h ) The left panel presents a stacked bar chart, whereas the right panel presents the individual data values in a bar chart format. Data are shown as mean ± SD (n = 3). Nuclei are labeled by DAPI (blue) staining. All scale bars, 50 μm.

Article Snippet: Membranes were blocked with 5 % nonfat milk in TBS-T (1 % Tween 20) overnight at 4 °C and incubated in the appropriate primary antibody in 3 % BSA in TBS-T for 2 h at room temperature shaking with PSAP (1:1,000, 10801-1-AP, Proteintech, USA), PGRN (1:1,000, AF 2557, R&D Systems, USA), and GAPDH (1:5000 60004-1-Ig, proteintech, USA).

Techniques: Expressing, Staining, Labeling

Journal: Biochemistry and Biophysics Reports

Article Title: Accumulation of prosaposin and progranulin around the subfornical organ induces polydipsia in SAP-D-deficient mice

doi: 10.1016/j.bbrep.2025.102388

Figure Lengend Snippet: Lysosomal localization of PSAP and PGRN expression around the SFO a) Immunofluorescence staining of LAMP1 (green) around the SFO in 10-month-old female WT and SAP-D −/− mice. Nuclei are labeled by DAPI (blue) staining. b) Quantification of LAMP1-stained areas in ( a ) relative to WT (%). The results of Student's t-tests for each panel are as follows: left panel, p = 0.0059, Cohen's d = 2.35 (95 % CI: 15.89, 67.66); and right panel: p = 0.0061, Cohen's d = 2.33 (95 % CI: 51.06, 220.89). Data are shown as the mean ± SD (n = 5). c) Triple immunofluorescent staining of PSAP (red), PGRN (green), and LAMP1 (cyan) around the SFO of 10-month-old female SAP-D −/− mice. Enlarged images indicated by the white squares (ⅰ-iv) were shown in c. d) Localization rate of PGRN or PSAP to LAMP1 in the SFO. The results of Student's t-tests for each panel are as follows: left panel, p = 0.0008, Cohen's d = 2.24 (95 % CI: -40.90, -13.73); and right panel, p = 0.2341, Cohen's d = 0.64 (95 % CI: 26.80, 7.17). Data are shown as the mean ± SD (n = 6 for WT-SFO and n = 8 for SAP-D −/− -SFO). e) Same experiment as in (c) on microglia/macrophage co-expressing PSAP and PGRN, or PGRN only, in the boundary and fornix regions of SAP-D −/− mice. Single image for each antibody was shown in white, while double or triple merged images were presented with red (PSAP or PGRN), green (LAMP1 or PGRN), or cyan (LAMP1) as indicated. and indicate the individual values in each group (b and d). ns: no significant difference. ∗∗∗ p < 0.001. ∗∗ p < 0.01. All scale bars, 10 μm.

Article Snippet: Membranes were blocked with 5 % nonfat milk in TBS-T (1 % Tween 20) overnight at 4 °C and incubated in the appropriate primary antibody in 3 % BSA in TBS-T for 2 h at room temperature shaking with PSAP (1:1,000, 10801-1-AP, Proteintech, USA), PGRN (1:1,000, AF 2557, R&D Systems, USA), and GAPDH (1:5000 60004-1-Ig, proteintech, USA).

Techniques: Expressing, Immunofluorescence, Staining, Labeling

Journal: Biochemistry and Biophysics Reports

Article Title: Accumulation of prosaposin and progranulin around the subfornical organ induces polydipsia in SAP-D-deficient mice

doi: 10.1016/j.bbrep.2025.102388

Figure Lengend Snippet: Increased protein levels of PSAP and PGRN within the SFO of SAP-D −/− mice a – b) , Coronal brain section at 0.7–0.8 mm posterior to bregma containing the SFO. a: DAPI staining. a-2: Enlarged view of the white square in a-1. SFO: subfornical organ, 3V: third ventricle. b; Double immunofluorescent staining of PSAP (red) and PGRN (green) in 10-month-old female WT and SAP-D −/− mice. DAPI (blue) staining showed the nuclei ( a and b ). Scale bar, 500 μm ( a and b ). White arrowheads indicate the SFO regions ( b ). c) Cerebral region from 3-, 6-, and 10-month-old male and female mice containing the SFO (0.7–0.8 mm posterior to ∗bregma) used for protein extraction and Western blot ( d – h ) using anti- PSAP, PGRN, and GAPDH antibodies. Quantification normalized to GAPDH expression and represented as the mean ± SD of three mice for each group. and indicate the individual values in each group ( e , f , h ). d – f) PSAP and PGRN protein levels in the SFO were remarkably increased. Their quantification by densitometric analysis is represented in e for male and f for female, respectively. e ) For PSAP/GAPDH, two-way ANOVA revealed a significant main effects of genotype (F(1,24) = 547.7, p < 0.0001, ηp 2 = 0.48, 95 % CI [−74.51, −62.43]), with no effect of age ( p = 0.46) or genotype × age interaction ( p = 0.47). Post-hoc Tukey's tests showed that SAP-D −/− differed from WT at 3 M ( p < 0.0001, Cohen's d = 9.99, 95 % CI [−86.30, −54.96]), 6 M ( p < 0.0001, Cohen's d = 7.24, 95 % CI [−79.04, −47.70]), and 10 M ( p < 0.0001, Cohen's d = 8.77, 95 % CI [−87.09, −55.75]). For PGRN/GAPDH, two-way ANOVA revealed a significant main effect of genotype (F(1,24) = 354.2, p < 0.0001, ηp 2 = 0.48, 95 % CI [−46.39, −37.22]), with no effect of age ( p = 0.73) or genotype × age interaction ( p = 0.76). Post-hoc Tukey's tests showed that SAP-D −/− differed from WT at 3 M ( p < 0.0001, Cohen's d = 7.87, 95 % CI [−52.71, −28.91]), 6 M ( p < 0.0001, Cohen's d = 5.35, 95 % CI [−52.82, −29.02]), and 10 M ( p < 0.0001, Cohen's d = 8.77, 95 % CI [−52.32, −28.52]). f ) For PSAP/GAPDH, two-way ANOVA revealed a significant main effect of genotype (F(1,24) = 611.1, p < 0.0001, ηp 2 = 0.48, 95 % CI [−74.42, −62.95]), with no effect of age ( p = 0.07) or genotype × age interaction (p = 0.07). Post-hoc Tukey's tests showed that SAP-D −/− differed from WT at 3 M ( p < 0.0001, Cohen's d = 9.99, 95 % CI [−86.30, −54.96]), 6 M ( p < 0.0001, Cohen's d = 7.24, 95 % CI [−79.04, −47.70]), and 10 M ( p < 0.0001, Cohen's d = 8.77, 95 % CI [−87.09, −55.75]). For PGRN/GAPDH, two-way ANOVA revealed a significant main effect of genotype (F(1,24) = 273.5, p < 0.0001, ηp 2 = 0.47, 95 % CI [−63.96, −49.76]), with no effect of age ( p = 0.71) or genotype × age interaction (p = 0.70). Post-hoc Tukey's tests showed that SAP-D −/− differed from WT at 3 M ( p < 0.0001, Cohen's d = 7.31, 95 % CI [−73.43, −36.60]), 6 M ( p < 0.0001, Cohen's d = 5.71, 95 % CI [−73.04, −36.22]), and 10 M ( p < 0.0001, Cohen's d = 5.35, 95 % CI [−73.08, −36.26]). g – h ) Comparison of PSAP and PGRN protein expression in the SFO, whole cerebrum, and cerebellum. The quantitative analysis is shown in h . h ) For PSAP/GAPDH, one-way ANOVA revealed a significant effect in SAP-D −/− mice (F(2,6) = 30.06, p = 0.0007, η 2 = 0.90), but not in WT mice ( p = 0.3461). Tukey's post hoc tests showed significant differences for SFO versus cerebellum ( p = 0.0010, Cohen's d = 5.26, 95 % CI [46.80, 119.1]) as well as and cerebrum versus cerebellum ( p = 0.0018, Cohen's d = 9.34, 95 % CI [38.39, 110.6]). There was no significant difference for SFO versus cerebrum ( p = 0.76, Cohen's d = 0.47, 95 % CI [−27.71, 44.54]). For PGRN/GAPDH, one-way ANOVA revealed a significant effect in SAP-D −/− mice (F(2,6) = 54.42, p = 0.0001, η 2 = 0.94), but not in WT mice ( p = 0.6327). Tukey's post hoc tests showed significant differences for SFO versus cerebrum ( p = 0.0005, Cohen's d = 5.43, 95 % CI [26.19, 57.84]) as well as and SFO versus cerebellum ( p = 0.0005, Cohen's d = 5.43, 95 % CI [26.19, 57.84]), but not for cerebrum versus cerebellum ( p = 0.3274, Cohen's d = 6.06, 95 % CI [−7.72, 23.92]). ns: no significant difference. ∗∗∗∗ p < 0.0001. ∗∗∗ p < 0.001. ∗∗ p < 0.01.

Article Snippet: The primary antibodies used were a rabbit antibody against PSAP (dilution 1:100, 10801-1-AP, Proteintech Group Inc., IL, USA), a sheep antibody against PGRN (dilution 1:100, AF 2557, R&D Systems Inc., MN, USA), a guinea pig antibody against c-Fos (dilution 1:500, 226308, Synaptic Systems GmbH, Göttingen, Germany), a rat antibody against CD68-FITC conjugated (dilution 1:500, MCA1957FA, BIO-RAD Laboratories Inc., CA, USA), and a rat antibody against LAMP1 (dilution 1:100, ab25245, Abcam, Cambridge, UK).

Techniques: Staining, Protein Extraction, Western Blot, Expressing, Comparison

Journal: Biochemistry and Biophysics Reports

Article Title: Accumulation of prosaposin and progranulin around the subfornical organ induces polydipsia in SAP-D-deficient mice

doi: 10.1016/j.bbrep.2025.102388

Figure Lengend Snippet: Increased PSAP and PGRN immunostaining in the SFO and its surrounding tissues in SAP-D −/− mice a) Double immunofluorescent staining of PSAP (red) and PGRN (green) around the SFO in 10-month-old female WT and SAP-D −/− mice. The white dotted lines enclose the SFO. b ) enlarged white ⅰ-iv squares in a, as indicated. White arrowheads indicate co-staining with anti-PGRN and PSAP antibodies. Open arrowheads indicate staining with PGRN alone. Nuclei are labeled by DAPI (blue) staining. All scale bars, 20 μm.

Article Snippet: The primary antibodies used were a rabbit antibody against PSAP (dilution 1:100, 10801-1-AP, Proteintech Group Inc., IL, USA), a sheep antibody against PGRN (dilution 1:100, AF 2557, R&D Systems Inc., MN, USA), a guinea pig antibody against c-Fos (dilution 1:500, 226308, Synaptic Systems GmbH, Göttingen, Germany), a rat antibody against CD68-FITC conjugated (dilution 1:500, MCA1957FA, BIO-RAD Laboratories Inc., CA, USA), and a rat antibody against LAMP1 (dilution 1:100, ab25245, Abcam, Cambridge, UK).

Techniques: Immunostaining, Staining, Labeling

Journal: Biochemistry and Biophysics Reports

Article Title: Accumulation of prosaposin and progranulin around the subfornical organ induces polydipsia in SAP-D-deficient mice

doi: 10.1016/j.bbrep.2025.102388

Figure Lengend Snippet: Infiltration of CD68-positive activated microglia/macrophages co-expressing PSAP and PGRN into the SFO and surrounding tissues a – b) Double immunofluorescent staining of PGRN (green) and CD68 (red) around the SFO in 10-month-old-female WT and SAP-D −/− mice. b , Magnified images of the indicated white squares in a . ⅰ: SFO, ⅱ: Fornix, and ⅲ: Perivascular, bv: blood vessel. c ) Quantification of PGRN- and/or CD68-staining in the SFO and surrounding areas in WT and SAP-D −/− mice. Data are shown as the mean ± SD (n = 3). The left panel presents a stacked bar chart, whereas the right panel shows the individual data values in a bar chart format. d – h ) Triple immunofluorescent staining of PSAP (red), PGRN (green), and CD68 (cyan) around the SFO in 10-month-old-female SAP-D −/− mice. e) Magnified images of the indicated white squares in d) ⅳ: Boundary, ⅴ: Fornix, and ⅵ: Perivascular. White arrowheads indicate triple co-staining with PSAP, PGRN, and CD68. Open arrowheads indicate PGRN signals alone. Co-localization rates of CD68 positive areas in PSAP ( f ), PGRN ( g ), and PSAP-PGRN staining areas ( h ) around the SFO of SAP-D −/− mice, respectively. f-h ) The left panel presents a stacked bar chart, whereas the right panel presents the individual data values in a bar chart format. Data are shown as mean ± SD (n = 3). Nuclei are labeled by DAPI (blue) staining. All scale bars, 50 μm.

Article Snippet: The primary antibodies used were a rabbit antibody against PSAP (dilution 1:100, 10801-1-AP, Proteintech Group Inc., IL, USA), a sheep antibody against PGRN (dilution 1:100, AF 2557, R&D Systems Inc., MN, USA), a guinea pig antibody against c-Fos (dilution 1:500, 226308, Synaptic Systems GmbH, Göttingen, Germany), a rat antibody against CD68-FITC conjugated (dilution 1:500, MCA1957FA, BIO-RAD Laboratories Inc., CA, USA), and a rat antibody against LAMP1 (dilution 1:100, ab25245, Abcam, Cambridge, UK).

Techniques: Expressing, Staining, Labeling

Journal: Biochemistry and Biophysics Reports

Article Title: Accumulation of prosaposin and progranulin around the subfornical organ induces polydipsia in SAP-D-deficient mice

doi: 10.1016/j.bbrep.2025.102388

Figure Lengend Snippet: Lysosomal localization of PSAP and PGRN expression around the SFO a) Immunofluorescence staining of LAMP1 (green) around the SFO in 10-month-old female WT and SAP-D −/− mice. Nuclei are labeled by DAPI (blue) staining. b) Quantification of LAMP1-stained areas in ( a ) relative to WT (%). The results of Student's t-tests for each panel are as follows: left panel, p = 0.0059, Cohen's d = 2.35 (95 % CI: 15.89, 67.66); and right panel: p = 0.0061, Cohen's d = 2.33 (95 % CI: 51.06, 220.89). Data are shown as the mean ± SD (n = 5). c) Triple immunofluorescent staining of PSAP (red), PGRN (green), and LAMP1 (cyan) around the SFO of 10-month-old female SAP-D −/− mice. Enlarged images indicated by the white squares (ⅰ-iv) were shown in c. d) Localization rate of PGRN or PSAP to LAMP1 in the SFO. The results of Student's t-tests for each panel are as follows: left panel, p = 0.0008, Cohen's d = 2.24 (95 % CI: -40.90, -13.73); and right panel, p = 0.2341, Cohen's d = 0.64 (95 % CI: 26.80, 7.17). Data are shown as the mean ± SD (n = 6 for WT-SFO and n = 8 for SAP-D −/− -SFO). e) Same experiment as in (c) on microglia/macrophage co-expressing PSAP and PGRN, or PGRN only, in the boundary and fornix regions of SAP-D −/− mice. Single image for each antibody was shown in white, while double or triple merged images were presented with red (PSAP or PGRN), green (LAMP1 or PGRN), or cyan (LAMP1) as indicated. and indicate the individual values in each group (b and d). ns: no significant difference. ∗∗∗ p < 0.001. ∗∗ p < 0.01. All scale bars, 10 μm.

Article Snippet: The primary antibodies used were a rabbit antibody against PSAP (dilution 1:100, 10801-1-AP, Proteintech Group Inc., IL, USA), a sheep antibody against PGRN (dilution 1:100, AF 2557, R&D Systems Inc., MN, USA), a guinea pig antibody against c-Fos (dilution 1:500, 226308, Synaptic Systems GmbH, Göttingen, Germany), a rat antibody against CD68-FITC conjugated (dilution 1:500, MCA1957FA, BIO-RAD Laboratories Inc., CA, USA), and a rat antibody against LAMP1 (dilution 1:100, ab25245, Abcam, Cambridge, UK).

Techniques: Expressing, Immunofluorescence, Staining, Labeling