progranulin antibody  (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: Journal of Neurochemistry

Article Title: Delivering Progranulin to Astrocytic Lysosomes Promotes Growth of Co‐Cultured Neurons

doi: 10.1111/jnc.70284

Figure Lengend Snippet: Recombinant progranulin stimulates dendritic outgrowth in primary hippocampal cultures. (A) Primary hippocampal cultures were plated onto coverslips, then subjected to media changes resulting in a final concentration of 0 or 10 μg/mL recombinant human progranulin. Cultures were maintained for a total of 4 days with progranulin levels replenished in a second media change to achieve final concentrations of 0 or 10 μg/mL recombinant human progranulin. (B) Neurons treated with recombinant progranulin had greater total dendritic length after 4 days in culture than neurons not treated with recombinant progranulin (linear mixed effects model, t (217) = 2.619, p = 0.00945, n = 105–116 neurons per group from three independent cultures). Violin and box plots represent the distribution of total dendritic length for all neurons analyzed, and dots represent the median length of all neurons from each culture. Representative images of MAP2 immunostaining are shown in (C) with 50 μm scale bars.

Article Snippet: The following primary antibodies were used for immunostaining: MAP2 (Thermo Fisher # PA1‐10005, RRID:AB_1076848), progranulin (R&D systems #AF2420, RRID:AB_2114489), GFAP (Agilent # Z0334, RRID:AB_10013382), S100β (Abcam #ab52642, RRID:AB_882426), HA tag (Cell Signaling Technologies #3724, RRID:AB_1549585), and Cathepsin D (R&D Systems #AF1029, RRID:AB_2087094).

Techniques: Recombinant, Concentration Assay, Immunostaining

Journal: Journal of Neurochemistry

Article Title: Delivering Progranulin to Astrocytic Lysosomes Promotes Growth of Co‐Cultured Neurons

doi: 10.1111/jnc.70284

Figure Lengend Snippet: Delivering progranulin to lysosomes stimulates dendritic outgrowth. (A) Primary hippocampal cultures were plated onto coverslips, then transduced with lentiviral vectors expressing GFP, progranulin (PGRN) or lysosome‐targeted progranulin (L‐PGRN) under the human PGK promoter. Cultures were maintained for 4 days before fixing and immunostaining. (B) The lenti‐PGK vectors transduced both neurons (MAP2+) and astrocytes (GFAP+), which were the two primary cell types observed in these cultures. (C) Virally expressed PGRN was secreted, while L‐PGRN exhibited no detectable secretion, even in media concentrated 5× using a centrifugal filter. (D, E) Both PGRN (linear mixed effects model, main effect of lentivirus, F (3,556) = 3.2966, p = 0.02024, PGRN versus GFP, p = 0.0404) and L‐PGRN (linear mixed effects model, p = 0.0119) stimulated dendritic outgrowth compared to GFP, while untransduced neurons exhibited comparable growth to those transduced with lenti‐GFP (linear mixed effects model, p = 0.9745). n = 124–159 neurons per group from 4 to 5 independent cultures (one culture did not include untransduced neurons). Violin and box plots represent the distribution of total dendritic length for all neurons analyzed, and dots represent the median length of all neurons from each culture. Scale bars in B and E represent 50 μm.

Article Snippet: The following primary antibodies were used for immunostaining: MAP2 (Thermo Fisher # PA1‐10005, RRID:AB_1076848), progranulin (R&D systems #AF2420, RRID:AB_2114489), GFAP (Agilent # Z0334, RRID:AB_10013382), S100β (Abcam #ab52642, RRID:AB_882426), HA tag (Cell Signaling Technologies #3724, RRID:AB_1549585), and Cathepsin D (R&D Systems #AF1029, RRID:AB_2087094).

Techniques: Transduction, Expressing, Immunostaining

Journal: Journal of Neurochemistry

Article Title: Delivering Progranulin to Astrocytic Lysosomes Promotes Growth of Co‐Cultured Neurons

doi: 10.1111/jnc.70284

Figure Lengend Snippet: Delivering progranulin to neuronal lysosomes does not stimulate dendritic outgrowth. (A) Primary hippocampal cultures were plated onto coverslips and transduced with hSyn‐IRES‐GFP vectors, with an empty IRES‐GFP vector serving as a control for the PGRN‐IRES‐GFP and L‐PGRN‐IRES‐GFP vectors. Cultures were maintained for 4 days before fixing and immunostaining. (B) The lenti‐hSyn vectors selectively transduced neurons (MAP2+ cells). (C) Virally expressed PGRN was secreted, while L‐PGRN exhibited no detectable secretion, even in media concentrated 5× using a centrifugal filter. (D, E) When administered at a viral dose that gave equivalent levels of PGRN as the lenti‐PGK vector, neither PGRN nor L‐PGRN (linear mixed effects model, main effect of lentivirus, F (3,328) = 1.5083, p = 0.2122) stimulated dendritic outgrowth compared to GFP. n = 76–89 neurons per group from 3 independent cultures. (F, G) When administered at a dose of equivalent viral copies, which produced higher levels of PGRN than the lenti‐PGK vector, PGRN stimulated dendritic outgrowth compared to GFP (linear mixed effects model, main effect of lentivirus, F (2,606) = 3.3301, p = 0.03644, PGRN vs. Ctrl, p = 0.0113), while L‐PGRN did not (linear mixed effects model, p = 0.3766). n = 202–207 neurons per group from 4 independent cultures. Violin and box plots represent the distribution of total dendritic length for all neurons analyzed, and dots represent median length of all neurons from each culture. Scale bars in B, E, and G represent 50 μm.

Article Snippet: The following primary antibodies were used for immunostaining: MAP2 (Thermo Fisher # PA1‐10005, RRID:AB_1076848), progranulin (R&D systems #AF2420, RRID:AB_2114489), GFAP (Agilent # Z0334, RRID:AB_10013382), S100β (Abcam #ab52642, RRID:AB_882426), HA tag (Cell Signaling Technologies #3724, RRID:AB_1549585), and Cathepsin D (R&D Systems #AF1029, RRID:AB_2087094).

Techniques: Transduction, Plasmid Preparation, Control, Immunostaining, Produced

Journal: Journal of Neurochemistry

Article Title: Delivering Progranulin to Astrocytic Lysosomes Promotes Growth of Co‐Cultured Neurons

doi: 10.1111/jnc.70284

Figure Lengend Snippet: Delivering progranulin to astrocytic lysosomes stimulates dendritic outgrowth. (A) Primary hippocampal cultures were plated onto coverslips and transduced with GFAP‐IRES‐GFP vectors, with the empty vector serving as a control for the PGRN‐IRES‐GFP and L‐PGRN‐IRES‐GFP vectors. Cultures were maintained for 4 days before fixing and immunostaining. (B) Lenti‐GFAP vectors selectively transduced astrocytes (GFAP+ cells). (C) Virally expressed PGRN was secreted, while L‐PGRN exhibited no detectable secretion, even in media concentrated 5× using a centrifugal filter. (D, E) Both PGRN (linear mixed effects model, main effect of lentivirus, F (4,801) = 5.4101, p = 0.0002662, PGRN vs. Ctrl, p < 0.0001) and L‐PGRN (linear mixed effects model, L‐PGRN vs. Ctrl, p = 0.00155) stimulated dendritic outgrowth compared to the control vector, while untransduced neurons did not have significantly different growth from those transduced with lenti‐IRES‐GFP (linear mixed effects model, p = 0.1539). As an additional control, this experiment included neurons transduced with lysosome‐targeted GFP (L‐GFP), which also did not stimulate growth relative to the control vector (linear mixed effects model, p = 0.1443). n = 139–193 neurons per group from 3 independent cultures. Violin and box plots represent the distribution of total dendritic length for all neurons analyzed, and dots represent the median length of all neurons from each culture. Scale bars in B and E represent 50 μm.

Article Snippet: The following primary antibodies were used for immunostaining: MAP2 (Thermo Fisher # PA1‐10005, RRID:AB_1076848), progranulin (R&D systems #AF2420, RRID:AB_2114489), GFAP (Agilent # Z0334, RRID:AB_10013382), S100β (Abcam #ab52642, RRID:AB_882426), HA tag (Cell Signaling Technologies #3724, RRID:AB_1549585), and Cathepsin D (R&D Systems #AF1029, RRID:AB_2087094).

Techniques: Transduction, Plasmid Preparation, Control, Immunostaining

Journal: Journal of Neurochemistry

Article Title: Delivering Progranulin to Astrocytic Lysosomes Promotes Growth of Co‐Cultured Neurons

doi: 10.1111/jnc.70284

Figure Lengend Snippet: Enhanced dendritic outgrowth in neurons co‐cultured with astrocytes transduced with L‐PGRN. (A) mature hippocampal astrocyte cultures were obtained by plating in DMEM with 10% serum and culturing for 1 week, then passaging to transwell inserts in serum‐free medium and transducing with lenti‐PGK vectors 4 days later. These transwell inserts containing mature astrocytes were placed over new primary hippocampal cultures, which were harvested after 4 days for immunostaining. Representative progranulin immunostaining in astrocytes is shown in (B). (C) PGRN was secreted by astrocytic cultures, while L‐PGRN was not. (D) Lack of progranulin secretion was confirmed by ELISA of conditioned media of several astrocyte cultures. Media from cultures transduced with lenti‐PGK‐GFP exhibited low background signal for human progranulin (hPGRN), which was dramatically increased in cultures transduced with lenti‐PGK‐PGRN (ANOVA main effect of lentivirus, F (2,12) = 6.974, p = 0.0098, PGRN versus GFP p = 0.0123 and PGRN vs. L‐PGRN p = 0.0297 by Tukey's post hoc test, n = 16 wells from 5 independent cultures). Cultures transduced with lenti‐PGK‐L‐PGRN did not differ from GFP ( p = 0.877 by Tukey's post hoc test) and also had dramatically less hPGRN in conditioned media than cultures transduced with lenti‐PGK‐PGRN ( p = 0.0297 by Tukey's post hoc test). (E) L‐PGRN was delivered to lysosomes of primary astrocytes, as shown by colocalization of the HA tag from virally expressed L‐PGRN with the lysosomal protease cathepsin D (CatD). (F, G) Despite the lack of progranulin secretion, neurons co‐cultured with astrocytes transduced with L‐PGRN had greater dendritic outgrowth compared to neurons cultured with GFP‐treated astrocytes (linear mixed effects model, main effect of lentivirus, F (2,580) = 7.6264, p = 0.0005379, L‐PGRN vs. GFP, p = 0.000619) while neurons co‐cultured with untransduced astrocytes had similar dendritic outgrowth as neurons cultured with GFP‐treated astrocytes (linear mixed effects model, p = 0.876719). n = 180–206 neurons per group from 4 independent cultures. Violin and box plots represent the distribution of total dendritic length for all neurons analyzed, and dots represent median length of all neurons from each culture. Scale bars represent 50 μm in B and G and 10 μm in E. Panel A created at Biorender.com , Arrant (2025) https://BioRender.com/cme038k .

Article Snippet: The following primary antibodies were used for immunostaining: MAP2 (Thermo Fisher # PA1‐10005, RRID:AB_1076848), progranulin (R&D systems #AF2420, RRID:AB_2114489), GFAP (Agilent # Z0334, RRID:AB_10013382), S100β (Abcam #ab52642, RRID:AB_882426), HA tag (Cell Signaling Technologies #3724, RRID:AB_1549585), and Cathepsin D (R&D Systems #AF1029, RRID:AB_2087094).

Techniques: Cell Culture, Transduction, Passaging, Immunostaining, Enzyme-linked Immunosorbent Assay

Journal: Journal of Neurochemistry

Article Title: Delivering Progranulin to Astrocytic Lysosomes Promotes Growth of Co‐Cultured Neurons

doi: 10.1111/jnc.70284

Figure Lengend Snippet: L‐PGRN reduces transcriptomic signatures of cellular reactivity in primary astrocytes. (A) To better understand how delivering progranulin to lysosomes changes the phenotype of primary astrocytes, we cultured mature astrocytes using the same procedure as previously used for neuronal co‐cultures. Instead of co‐culturing with neurons, we switched astrocytes to a “co‐culture medium” identical to that used in co‐cultures. After 4 days in co‐culture medium, we harvested RNA from these astrocytes and conducted bulk RNA sequencing. (B) Nearly all cells in these cultures were immunoreactive for the astrocytic markers GFAP and S100β, and (C) bulk RNA sequencing confirmed the enrichment of astrocyte marker genes versus other cell types. (D) Gene set enrichment analysis of sequencing data using Hallmark pathways suggested potential increases in proliferation and decreases in cellular reactivity. n = 4 samples per treatment from 4 independent cultures. Panel A created at Biorender.com , Arrant (2025) https://BioRender.com/560o6wr . Raw counts of genes in panel C are provided in Table , and a full list of enriched Hallmark pathways from panel D is provided in Table .

Article Snippet: The following primary antibodies were used for immunostaining: MAP2 (Thermo Fisher # PA1‐10005, RRID:AB_1076848), progranulin (R&D systems #AF2420, RRID:AB_2114489), GFAP (Agilent # Z0334, RRID:AB_10013382), S100β (Abcam #ab52642, RRID:AB_882426), HA tag (Cell Signaling Technologies #3724, RRID:AB_1549585), and Cathepsin D (R&D Systems #AF1029, RRID:AB_2087094).

Techniques: Cell Culture, Co-Culture Assay, RNA Sequencing, Marker, Sequencing

Journal: Journal of Neurochemistry

Article Title: Delivering Progranulin to Astrocytic Lysosomes Promotes Growth of Co‐Cultured Neurons

doi: 10.1111/jnc.70284

Figure Lengend Snippet: Reducing the number of astrocytes occludes the pro‐growth effects of L‐PGRN. (A) Primary hippocampal cultures were plated onto coverslips, transduced with lenti‐PGK‐GFP or L‐PGRN, then treated with 5 μM Ara‐C approximately 24 h after plating. Cultures were maintained for a total of 4 days. (B) Ara‐C treatment modestly reduced the number of neurons in these cultures (linear mixed effects model main effect of Ara‐C, F (1,108) = 16.5707, p < 0.0001, ** = p = 0.00122 for L‐PGRN vs. L‐PGRN+Ara‐C by Tukey's post hoc test). L‐PGRN–treated cultures treated with Ara‐C also had slightly fewer neurons per visual field than GFP‐treated cultures treated with Ara‐C (linear mixed effects model main effect of lentivirus, F (1,108) = 6.3728, p = 0.01304, * = p = 0.04435 for GFP + Ara‐C vs. L‐PGRN+Ara‐C by Tukey's post hoc test), though there was not a significant difference between lentiviral groups under control conditions ( p = 0.65663 by Tukey's post hoc test). (C) Ara‐C nearly completely eliminated astrocytes from these cultures (linear mixed effects model main effect of Ara‐C, F (1,108) = 74.08, p < 0.0001, *** = p < 0.001 for GFP vs. GFP + Ara‐C and L‐PGRN vs. L‐PGRN+Ara‐C by Tukey's post hoc test). There was not a significant difference between lentiviral treatment groups in the number of astrocytes per visual field (linear mixed effects model main effect of lentivirus, F (1,108) = 0.707, p = 0.4023). n = 28–29 coverslips from four independent cultures. Violin and box plots represent the distribution of cell count for all coverslips analyzed, and dots represent median cell count from each culture. (D, E) L‐PGRN+Ara‐C wells expressed less progranulin than L‐PGRN + vehicle wells by immunoblot (paired t ‐test, t (2) = 4.403, p = 0.0479, n = 10–11 replicates from 3 independent cultures). (F, G) However, analysis of progranulin immunoreactivity in neurons revealed a non‐significant trend for lower progranulin (paired t test, t (3) = 2.237, p = 0.1113, n = 12 replicates from 4 independent cultures). Scale bars in G represent 100 μm. (H) Analysis of dendritic length in vehicle‐treated cultures replicated our prior finding that L‐PGRN promotes dendritic outgrowth (linear mixed effects model, main effect of lentivirus, F (1,996) = 5.1756, p = 0.02312, ** = p = 0.00802 for GFP vs. L‐PGRN by Tukey's post hoc test), but also revealed that Ara‐C treatment stimulated dendritic outgrowth (linear mixed effects model main effect of Ara‐C, F (1,996) = 15.5234, p < 0.0001, *** = p < 0.001 for GFP vs. GFP + Ara‐C by Tukey's post hoc test) and that among Ara‐C treated neurons, those transduced with L‐PGRN did not have significantly different dendritic growth from those transduced with GFP (linear mixed effects model, p = 0.99088). n = 215–275 neurons per group from 4 independent cultures. Violin and box plots represent the distribution of total dendritic length for all neurons analyzed, and dots represent median length of all neurons from each culture. Representative images of MAP2 immunostaining are shown in (I) with 50 μm scale bars.

Article Snippet: The following primary antibodies were used for immunostaining: MAP2 (Thermo Fisher # PA1‐10005, RRID:AB_1076848), progranulin (R&D systems #AF2420, RRID:AB_2114489), GFAP (Agilent # Z0334, RRID:AB_10013382), S100β (Abcam #ab52642, RRID:AB_882426), HA tag (Cell Signaling Technologies #3724, RRID:AB_1549585), and Cathepsin D (R&D Systems #AF1029, RRID:AB_2087094).

Techniques: Transduction, Control, Cell Counting, Western Blot, Immunostaining

Journal: Neurobiology of disease

Article Title: Reduction of sphingomyelinase activity associated with progranulin deficiency and frontotemporal dementia

doi: 10.1016/j.nbd.2025.107024

Figure Lengend Snippet: Progranulin interacts with acid sphingomyelinase but not neutral sphingomyelinase 2. A , HEK293T cells were transfected with HA-GRN (GRN) and either ASMase-FLAG (ASM) or nSMase2-FLAG (nSM2), then immunoprecipitated with anti-HA. Co-immunoprecipitation of ASMase-FLAG, but not nSMase2-FLAG, indicates progranulin binding with ASMase but not nSMase2. Full-length blot in . B-E , Proximity ligation assay in GRN -WT and GRN -KO HEK293 cells transfected with ASMase-FLAG or nSMase-FLAG, using anti-progranulin (endogenous) and anti-FLAG antibodies. There were numerous PLA puncta with GRN-ASMase but not with GRN-nSMase2. GRN -WT, progranulin wildtype HEK293 cells; GRN -KO, progranulin knockout HEK293 cells. Scale bar = 10 μm.

Article Snippet: Cells were probed overnight with primary antibodies to human progranulin (0.04 μg/mL, goat polyclonal, R&D systems #AF2420, RRID: AB_2114489) and DYKDDDDK (FLAG) tag (1:25000, rabbit monoclonal, D6W5B, Cell Signaling Technology, RRID: AB_2572291) in 5 % bovine serum albumin in 1× PBS.

Techniques: Transfection, Immunoprecipitation, Binding Assay, Proximity Ligation Assay, Knock-Out

Journal: Neurobiology of disease

Article Title: Reduction of sphingomyelinase activity associated with progranulin deficiency and frontotemporal dementia

doi: 10.1016/j.nbd.2025.107024

Figure Lengend Snippet: Post-transcriptional loss of nSMase2 in the frontal cortex of progranulin mutation–positive and –negative FTLD-TDP-A. A-B , Western blot for nSMase2 in the frontal cortex reveals loss of nSMase2 in FTD- GRN and S-TDP-A cases (ANOVA, F (4,33) = 5.036, p = 0.0028, Tukey’s multiple comparisons * p < 0.05). C , RT-qPCR from frontal cortex for SMPD3 , the gene encoding nSMase2, reveals no change in RNA in any experimental group (ANOVA, F (4,33) = 1.779, p = 0.1566). FTD- GRN , GRN mutation-positive FTLD-TDP-A; S-TDP-A, sporadic FTLD-TDP-A; S-TDP-C, sporadic FTLD-TDP-C; Pick, Pick’s disease. B is a cropped image from a representative blot; the full-length representative blot is in .

Article Snippet: Cells were probed overnight with primary antibodies to human progranulin (0.04 μg/mL, goat polyclonal, R&D systems #AF2420, RRID: AB_2114489) and DYKDDDDK (FLAG) tag (1:25000, rabbit monoclonal, D6W5B, Cell Signaling Technology, RRID: AB_2572291) in 5 % bovine serum albumin in 1× PBS.

Techniques: Mutagenesis, Western Blot, Quantitative RT-PCR