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 GRN knockout (KO) in iNeurons leads to accumulation of TMEM106B C-terminal fragments (CTFs) in lysosomes. Purified lysosomes (LysoIP; top) and whole cell lysates (bottom) were from GRN wild type (WT) iNeurons without LysoTag (C-terminally tagged TMEM192-3xHA), GRN WT iNeurons with LysoTag, and GRN KO iNeurons with LysoTag. Lysosomes were purified by immunoprecipitation using the LysoTag. 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 (Beta-tubulin for whole cell lysates; LAMP1 for purified lysosomes), then normalizing to the first bar (WT with LysoTag for purified lysosomes or WT without LysoTag for whole cell lysates). C. qRT-PCR analysis shows that GRN KO does not alter TMEM106B mRNA levels in iNeurons. D. Western blots show that Grn KO in mice leads to accumulation of Tmem106b CTFs in lysosomes. Purified lysosomes were from livers of 6-month-old Grn WT mice without LysoTag, Grn WT mice with LysoTag, and Grn KO mice with LysoTag. Lysosomes were purified by immunoprecipitation using the LysoTag. E. Quantification of Tmem106b dimers and CTFs from panel D. Normalized ratios were calculated by dividing the intensity of each Tmem106b species (dimer or CTF) by the loading control (LAMP1), then normalizing to that of Grn WT with LysoTag. Bar plots represent the mean, and each dot represents a replicate (n = 3-4 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, Knock-Out, Purification, Immunoprecipitation, Control, Quantitative RT-PCR

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: Cells

Article Title: Acute Cold Exposure Cell-Autonomously Reduces mTORC1 Signaling and Protein Synthesis Independent of AMPK

doi: 10.3390/cells15010065

Figure Lengend Snippet: Acute cold exposure downregulates protein synthesis in an AMPK-independent manner. Wild-type (WT) and AMPK double-knockout (dKO) myoblasts [ n = 3 biological replicates (from separate cultures)/group] were incubated at 37 °C or 26 °C for 1 h, with the addition of puromycin for the final 30 min of incubation. The cells were then harvested and assayed for indicators of AMPK activity, anabolic signaling through mTORC1, and protein synthesis by Western blotting. ( A ) Levels of phosphorylated ACC (Ser79/212; p-ACC); ( B ) levels of phosphorylated p70S6k (Thr389; p-p70S6k); ( C ) levels of phosphorylated S6 (Ser240/244; p-rpS6); ( D ) levels of puromycin incorporation (indicative of protein synthesis); ( E ) representative blots for phosphorylated (p-ACC, p-p70S6k, p-rps6) and total (t-ACC, t-p70S6k, t-rpS6, puromycin) proteins shown in ( A – D ), and representative Ponceau stain for total protein showing equal protein loading and effective transfer. Data are means +/− S.E.M. Significant differences identified by 2 × 2 factorial ANOVA are indicated on the graph. Where only main effects without an interaction were detected, a = main effect of temperature; b = main effect of genotype. Where a significant interaction was detected, Bonferroni post hoc analysis was performed to identify significant differences between individual groups: * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001; ns = no significant difference.

Article Snippet: Primary antibodies [Puromycin (Millipore MABE343, Bedford, MA, USA), phospho-ACC (Cell Signaling #3661, Danvers, MA, USA), ACC (Cell Signaling #3676), phospho-S6K (Cell Signaling #9234), S6K (Cell Signaling #2708), phospho-S6 (Cell Signaling #4858), S6 (Cell Signaling #2217)] were applied for either 4 h at room temperature or overnight at 4 °C.

Techniques: Double Knockout, Incubation, Activity Assay, Western Blot, Staining

Journal: Open biology

Article Title: Kaiso mediates transcription and RNA splicing in colorectal carcinoma: role of BRCA1 in the Kaiso enhanceosome.

doi: 10.1098/rsob.240329

Figure Lengend Snippet: Figure 1. Visual inspection of VDA3 and GRB2 promoters (ENCODE ChIP-seq profile) (lower panels) and bioinformatic profiles (upper panels right) of top 15 ranked eKBS co-occupants for BRCA1 and ZBTB33/Kaiso across ENCODE data across multiple cancer cell lines. (A) Diagram of the Kaiso domains. (B) Co-occupants of BRCA1 and Kaiso/ZBTB33 are rank ordered by Z score. Purple fill mark top core hits. Light grey mark hits common to both. (C) The location of the eKBS in the VDA3 and GRB2 promoters is indicated by the ZBTB33/Kaiso peak; the BRCA1 and CHD2 peaks are highlighted with red arrows. eKBS, encode-derived Kaiso-binding site.

Article Snippet: Immunoprecipitations of crosslinked DNA/proteins were performed with BRCA1 antibody (Cell Signaling, cat. no. 9010) or IgG control antibody of the same class.

Techniques: ChIP-sequencing, Derivative Assay, Binding Assay

Journal: Open biology

Article Title: Kaiso mediates transcription and RNA splicing in colorectal carcinoma: role of BRCA1 in the Kaiso enhanceosome.

doi: 10.1098/rsob.240329

Figure Lengend Snippet: Figure 2. Kaiso and BRCA1 interact directly. Left three panels, BRCA1 and Kaiso interact specifically by PLA—the interaction takes place primarily but not exclusively in the nucleus. M, mouse, R, rabbit. Right panels: DNA damage causes striking translocation of Kaiso into the nucleus. Detection is by a single antibody variation of PLA. Each dot represents one molecule of Kaiso.

Article Snippet: Immunoprecipitations of crosslinked DNA/proteins were performed with BRCA1 antibody (Cell Signaling, cat. no. 9010) or IgG control antibody of the same class.

Techniques: Translocation Assay

Journal: Open biology

Article Title: Kaiso mediates transcription and RNA splicing in colorectal carcinoma: role of BRCA1 in the Kaiso enhanceosome.

doi: 10.1098/rsob.240329

Figure Lengend Snippet: Figure 5. ChIP-seq analysis of Kaiso and BRCA1 DNA-binding sites. (A) Number of gene promoters, exons, introns or intergenic regions bound by Kaiso or BRCA1 antibody in HCT116 cells. (B) Distribution of Kaiso and BRCA1 antibody binding to various regions of the genes. (C) sVenn diagram visualizing the gene promoters with or without overlap of BRCA1 and Kaiso antibody binding.

Article Snippet: Immunoprecipitations of crosslinked DNA/proteins were performed with BRCA1 antibody (Cell Signaling, cat. no. 9010) or IgG control antibody of the same class.

Techniques: ChIP-sequencing, Binding Assay

Journal: Open biology

Article Title: Kaiso mediates transcription and RNA splicing in colorectal carcinoma: role of BRCA1 in the Kaiso enhanceosome.

doi: 10.1098/rsob.240329

Figure Lengend Snippet: Figure 4. (A) Encode data showing how CHD2, BRCA1, ZBT33 and ETS binding sites overlap on the CCNC promoter. (B) BRCA1 transactivates Kaiso as eKBS sites. The CCNC promoter was subcloned into the ‘empty’ vector PGL3basic and assayed for in vitro transcriptional activity after transient transfection of the empty vector (lanes 1−4) or the same vector expressing the constructs indicated across the bottom. (C) As in (B), but the experimental constructs compare the effects of endogenous Kaiso (lane 4), to transiently overexpressed Kaiso (lane 5) and a dominant negative (DN) Kaiso (lacking N-terminal BTB domain; lane 6). BRCA1 elevates transcription compared with Kaiso overexpression alone by 30%. DN-Kaiso suppresses transcription relative to endogenous Kaiso alone.

Article Snippet: Immunoprecipitations of crosslinked DNA/proteins were performed with BRCA1 antibody (Cell Signaling, cat. no. 9010) or IgG control antibody of the same class.

Techniques: Binding Assay, Plasmid Preparation, In Vitro, Activity Assay, Transfection, Expressing, Construct, Dominant Negative Mutation, Over Expression

Journal: Open biology

Article Title: Kaiso mediates transcription and RNA splicing in colorectal carcinoma: role of BRCA1 in the Kaiso enhanceosome.

doi: 10.1098/rsob.240329

Figure Lengend Snippet: Figure 6. Schematic showing hypothetical interactions of eKBS co-occupants in proximal promoter transcriptional elongation. Components of the complex are anchored to DNA via Kaiso, which binds directly to the TCTCGCGAGA motif (aka the eKBS) near the transcriptional start site (TSS). BRCA1 is recruited to the CHD4/Kaiso complex, RNA Polymerase II+GTFs bind to BRCA1 at the TATA box of a promoter, and transcription is activated with the help of INRs.

Article Snippet: Immunoprecipitations of crosslinked DNA/proteins were performed with BRCA1 antibody (Cell Signaling, cat. no. 9010) or IgG control antibody of the same class.

Techniques:

Journal: Open biology

Article Title: Kaiso mediates transcription and RNA splicing in colorectal carcinoma: role of BRCA1 in the Kaiso enhanceosome.

doi: 10.1098/rsob.240329

Figure Lengend Snippet: Figure 9. (A) Bar graph of enriched terms across input gene lists that bind both BRCA1 and Kaiso in HCT116 cells, coloured by p-value. (B) Summary of enrichment analysis in transcription factor targets from analysis of genes binding both BRCA1 and Kaiso in HCT116 cells. (C) Summary of enrichment analysis in transcriptional regulatory relationships unraveled by sentence-based text mining (TRRUST) analysis of genes binding both BRCA1 And Kaiso in HCT116 cells.

Article Snippet: Immunoprecipitations of crosslinked DNA/proteins were performed with BRCA1 antibody (Cell Signaling, cat. no. 9010) or IgG control antibody of the same class.

Techniques: Binding Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: SHIP-1 Couples to the Dectin-1 hemITAM and Selectively Modulates Reactive Oxygen Species Production in Dendritic Cells in Response to Candida albicans.

doi: 10.4049/jimmunol.1402874

Figure Lengend Snippet: FIGURE 6. Syk, PI3K, and PDK1 mediate enhanced NADPH oxidase–triggered ROS production in SHIP-1–deficient GM-BM. Purified GM-BM from WT and CD11cDSHIP-1 mice were pretreated for 30 min with 3 mM of the Syk inhibitor R-406 (A), 10 mM of the ERK inhibitor UO126 (B), 25 mM of the PI3K inhibitor Ly294002 (C), 0.75 mM Akt inhibitor VIII (D), 1 mM of the PDK1 inhibitor BX-795 (E), or 5 mM of the NADPH oxidase inhibitor DPI (F) prior to stimulation with HKC at a 1:10 ratio. ROS production was monitored by chemiluminescence during the indicated times and expressed as relative light units per second (RLU/s). Left graphs show a representative experiment. Right histograms show arithmetic mean + SEM of the maximal ROS production corresponding to three to four independent experiments. (G) Purified GM-BM from WT and CD11cDSHIP-1 mice were exposed to HKC for the indicated times, and phospho-p40phox (P-p40phox) was detected by Western blot. A representative experiment of three is shown (left panel). Densitometric analysis of P-p40phox relative to b-actin was conducted (right panel). Bars depict arithmetic mean + SEM from three independent experiments. *p , 0.05, **p , 0.01, ***p , 0.001, one-way ANOVA.

Article Snippet: Samples were run on Mini-PROTEAN TGX PRECAST Gels (Bio-Rad, Hercules, CA) and transferred to a nitrocellulose membrane (Bio-Rad) for blotting with Abs against b-actin (C4) from Santa Cruz, and P-Syk (C87C1), P-ERK (E10), ERK (9102s), P-p38 (28B10), p38 (9212s), P-p40phox (4311s), IkBa (9242s), P-Akt (9275s), and Akt (40D4) from Cell Signaling.

Techniques: Western Blot

Journal: Molecular medicine reports

Article Title: VHL loss predicts response to Aurora kinase A inhibitor in renal cell carcinoma cells.

doi: 10.3892/mmr.2018.9038

Figure Lengend Snippet: Figure 2. 769‑P cells with VHL re‑expression show resistance to alisertib. (A) VHL and AURKA protein expression in 769‑P cells transfected with control or VHL plasmid was analyzed by immunoblotting. GAPDH was employed as a loading control. (B) The anti‑proliferative activity of alisertib against cells was assessed by Cell Counting Kit ‑8 assay. *P<0.01 vs. vector. VHL, von Hippel‑Lindau tumor suppressor; AURKA, Aurora kinase A; IC50, half‑maximal inhibitory concentration.

Article Snippet: Appropriate antibodies against VHL (1:1,000), AURKA (1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA), GAPDH (1:5,000; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) were used.

Techniques: Expressing, Transfection, Control, Plasmid Preparation, Western Blot, Activity Assay, Cell Counting, Concentration Assay

Journal: Molecular medicine reports

Article Title: VHL loss predicts response to Aurora kinase A inhibitor in renal cell carcinoma cells.

doi: 10.3892/mmr.2018.9038

Figure Lengend Snippet: Figure 1. VHL expression profiles and alisertib anti‑proliferative activities in RCC cells. (A) VHL and AURKA protein expression was detected by immunob lotting. GAPDH was employed as a loading control. (B) Anti‑proliferative activity was assessed by Cell Counting Kit‑8 assay following exposure to alisertib for 72 h. (C) Alisertib anti‑tumor activity in vivo. VHL, von Hippel‑Lindau tumor suppressor; RCC, renal cell carcinoma; AURKA, Aurora kinase A; IC50, half‑maximal inhibitory concentration; TGI, tumor growth inhibition rate.

Article Snippet: Appropriate antibodies against VHL (1:1,000), AURKA (1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA), GAPDH (1:5,000; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) were used.

Techniques: Expressing, Control, Activity Assay, CCK-8 Assay, In Vivo, Concentration Assay, Inhibition

Journal: Molecular medicine reports

Article Title: VHL loss predicts response to Aurora kinase A inhibitor in renal cell carcinoma cells.

doi: 10.3892/mmr.2018.9038

Figure Lengend Snippet: Figure 4. VHL regulates AURKA levels via HIF‑dependent and ‑independent pathways. (A) Hypoxia could induce AURKA protein upregulation upon analysis of HIF‑1 and AURKA protein expression in CAK‑I cells by immunoblotting; GAPDH was employed as a loading control. (B) The 26S proteasome inhibitor MG132 (10 µM) rescued the downregulation of AURKA in 769‑P cells re‑expressed with pVHL. *P<0.01 vs. empty vector. (C) pVHL promoted AURKA degradation. 769‑p cells were transfected with either pVHL or vector plasmid. Cells were treated with 100 µg/ml CHX and harvested at the indicated time points, and cell lysates were prepared. Proteins from cell lysates were subjected to western blotting with anti‑AURKA and anti‑GAPDH antibodies. Relative protein levels were plotted from the integrated optical density of the AURKA bands on the western blot (lower panel). VHL, von Hippel‑Lindau tumor suppressor; AURKA, Aurora kinase A; IC50, half‑maximal inhibitory concentration; HIF, hypoxia inducing factor; pVHL, VHL protein; CHX, cycloheximide.

Article Snippet: Appropriate antibodies against VHL (1:1,000), AURKA (1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA), GAPDH (1:5,000; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) were used.

Techniques: Expressing, Western Blot, Control, Plasmid Preparation, Transfection, Concentration Assay

Journal: Molecular medicine reports

Article Title: VHL loss predicts response to Aurora kinase A inhibitor in renal cell carcinoma cells.

doi: 10.3892/mmr.2018.9038

Figure Lengend Snippet: Figure 3. shRNA knockdown of VHL in CAKI cells confers decreased alisertib sensitivity. (A) VHL and AURKA protein expression in CAKI cells transfected with control or VHL shRNA was analyzed by immunoblotting. GAPDH was employed as a loading control. (B) The anti‑proliferative activity of alisertib against cells was assessed by Cell Counting Kit ‑8 assay. *P<0.01 vs. vector. (C) Inhibition of tumor growth by alisertib in xenografts of CAKI cells transfected with control or VHL shRNA. VHL, von Hippel‑Lindau tumor suppressor; AURKA, Aurora kinase A; IC50, half‑maximal inhibitory concentration; shRNA, short hairpin RNA.

Article Snippet: Appropriate antibodies against VHL (1:1,000), AURKA (1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA), GAPDH (1:5,000; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) were used.

Techniques: shRNA, Knockdown, Expressing, Transfection, Control, Western Blot, Activity Assay, Cell Counting, Plasmid Preparation, Inhibition, Concentration Assay