tau microtubule binding domain  (R&D Systems)

Journal: bioRxiv

Article Title: Septin crosstalk with microtubules and actin is regulated by a GSK3-dependent phosphoswitch

doi: 10.64898/2026.05.06.723191

Figure Lengend Snippet: (A) Total internal reflection fluorescence (TIRF) microscopy images of taxol-stabilized microtubules (magenta) following incubation with 1 nM GFP-SEPT9_i1 (green) for wild type, and S82A/S85A, and S82E/S85E mutants. Scale bars, 10 μm. (B) Quantification of mean GFP fluorescence intensity per microtubule length following incubation with 1 nM GFP-SEPT9_i1 ( n = 30; WT, S82A/S85A, S82E/S85E). Pairwise comparisons were statistically analyzed using an unpaired Welch’s t-test. *, p < 0.05; ****, p < 0.0001 (C) TIRF microscopy images of taxol-stabilized microtubules (magenta) following incubation with 1 nM of the indicated msGFP-tagged wild-type SEPT2/6/7/9_i1, SEPT2/6/7/9_i1-S82A/S82A, and msGFP-SEPT2/6/7/9_i1-S82E/S85E (green). Scale bars, 10 μm. (D) Quantification of mean GFP fluorescence intensity per microtubule length following incubation with 1 nM wild-type GFP-SEPT9_i1 ( n = 30), msGFP-SEPT2/6/7/9_i1-S82A/S82A ( n = 30), and msGFP-SEPT2/6/7/9_i1-S82E/S85E ( n = 30). Pairwise comparisons were statistically analyzed using a Mann-Whitney U-test. ****, p < 0.0001 All plots show mean ± SEM.

Article Snippet: Taxol-stabilized microtubules were prepared by incubating unlabeled (80%), HiLyte647 (10%) and biotin conjugated (10%) porcine brain tubulin (Cytoskeleton Inc) in BRB80 (80 mM PIPES pH 6.9, 1 mM EGTA pH 6.9, 2 mM MgCl2, 10% (v/v) glycerol) supplemented with 1 mM GTP at 37°C for 35 minutes.

Techniques: Fluorescence, Microscopy, Incubation, MANN-WHITNEY

Journal: Journal of Sport and Health Science

Article Title: Weightlifting outperforms voluntary wheel running for improving adiposity and insulin sensitivity in obese mice

doi: 10.1016/j.jshs.2025.101100

Figure Lengend Snippet: Resistance exercise exceeds the benefits of endurance exercise in ameliorating metabolic dysfunction. Following 8 weeks of diet and exercise interventions, all mice were assessed for their metabolic function by GTT, ITT, and skeletal muscle response of Akt and AS160 phosphorylation to injection of insulin measured by Western blot. (A–C) HOMA-IR taken after an overnight fast for baseline glucose and insulin. (D and E) GTT from 0–120 min and calculated AUC; colored * indicates significant difference from NC-SED. (F and G) ITT from 0–60 min and calculated AUC; colored * indicates significant difference from NC-SED. (H–N) pAkt stimulation, AS160 S318, and AS160 T642 in hindlimb muscles before and after insulin injection and the pre–post ∆ in phosphorylation. (O and P) Total and phosphorylated 4E-BP1. (Q–T) Western results for Raptor, COX4, LC3 II/I, and ubiquitin staining. Representative western blot images inset right. Data presented as mean ± standard error of the mean. Statistical analysis performed by analysis of variance between groups: * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. NC-SED n : 8–16 (white); HFD-SED n : 8–18 (red); HFD-R EX n : 8–16 (blue); HFD-E EX n : 8–15 (green). 4E-BP1 = Eukaryotic translation initiation factor 4E binding protein; Akt = protein kinase B; AS160 = Akt substrate 160; COX4 = cytochrome c oxidase 4; CS = citrate sythase; E EX = endurance exercise; GAPDH = glyceraldehyde 3 phosphate dehydrogenase; GTT = glucose tolerance test; HFD = high fat diet; HOMA-IR = homeostatic model assessment for insulin resistance; iAUC = integrated area under the curve; ITT = insulin tolerance test; LC3 II/I = microtubule-associated protein light chain 3; NC = normal chow; pAkt = phospho-Akt; R EX = resistance exercise; SED = sedentary; Ub = ubiquitin.

Article Snippet: Primary antibodies used for analysis were from Cell Signaling Technologies (Danvers, MA, USA) and diluted 1:1000 unless otherwise stated as follows: protein kinase B (Akt; 1:500; #4691; Cell Signaling Technologies), phospho-Akt (pAkt) S473 (1:500; #9271; Cell Signaling Technologies), Ubiquitin (#3933; Cell Signaling Technologies), microtubule-associated protein light chain 3 (LC3 II/I; #4018; Cell Signaling Technologies), cytochrome c oxidase subunit 4(COX4; #11967; Cell Signaling Technologies), Akt substrate 160 (AS160 S318; #8619; Cell Signaling Technologies), AS160 T642 (#8881; Cell Signaling Technologies), eukaryotic translation initiation factor 4E binding protein (4E-BP1; #9452; Cell Signaling Technologies), and glyceraldehyde 3 phosphate dehydrogenase (GAPDH; #2118; Cell Signaling Technologies).

Techniques: Phospho-proteomics, Injection, Western Blot, Muscles, Ubiquitin Proteomics, Staining, Binding Assay

Journal: Precision Clinical Medicine

Article Title: Agrimol B inhibits pancreatic ductal adenocarcinoma by induction of lethal mitophagy through decreasing mitochondrial transcription termination factor 3

doi: 10.1093/pcmedi/pbag009

Figure Lengend Snippet: Agrimol B induces PINK1/Parkin pathway-dependent mitophagy initiation in PDAC cells. (A) Western blot analysis of PINK1, Parkin, and LC3 in the mitochondria of PANC-1 and AsPC-1 cells. (B) Western blot analysis of Parkin in the mitochondria and cytoplasm of PANC-1 and AsPC-1 cells. (C) Western blot analysis of LC3 in the presence or absence of Agrimol B in the presence or absence of Mdivi-1 for 24 h. (D, E) Western blot analysis of LC3 in PDAC cells transfected with siScramble, siPINK1, or siParkin following treatment with or without Agrimol B. (F) Western blot analysis of LC3 in PANC-1 and AsPC-1 cells with or without Agrimol B in the presence or absence of wortmannin. (G-I) Immunofluorescence analysis of LC3 in PDAC cells treated with or without Agrimol B in the presence or absence of wortmannin. Scale bars, 10 μm.

Article Snippet: An anti-microtubule-associated protein light chain 3 (LC3) antibody (NB100-2220) was purchased from Novus, while anti-MTERF3 (EM1701-29) and lysosomal associated membrane protein 2 (LAMP2) (M1603-5) antibodies were purchased from HuaBio.

Techniques: Western Blot, Transfection, Immunofluorescence

Journal: Precision Clinical Medicine

Article Title: Agrimol B inhibits pancreatic ductal adenocarcinoma by induction of lethal mitophagy through decreasing mitochondrial transcription termination factor 3

doi: 10.1093/pcmedi/pbag009

Figure Lengend Snippet: Agrimol B blocks autophagic flux in PDAC cells. (A, B) Western blot analysis of P62 and CTSD in PANC-1 and AsPC-1 cells treated with Agrimol B for 24 h. (C, E, F) Immunofluorescence analysis of RFP-GFP-LC3 after PANC-1 and AsPC-1 cells were transfected with RFP-GFP-LC3 for 48 h, followed by treatment with or without Agrimol B for another 24 h. Scale bars, 10 μm. (D, G-L) Immunofluorescence analysis of the colocalization of endogenous LC3 with LAMP2 after treatment with Agrimol B or rapamycin for 24 h in PANC-1 and AsPC-1 cells. Scale bars, 10 μm. (M-O) Immunofluorescence analysis of LC3 in PDAC cells treated with or without Agrimol B in the presence or absence of HCQ. Scale bars, 10 μm.

Article Snippet: An anti-microtubule-associated protein light chain 3 (LC3) antibody (NB100-2220) was purchased from Novus, while anti-MTERF3 (EM1701-29) and lysosomal associated membrane protein 2 (LAMP2) (M1603-5) antibodies were purchased from HuaBio.

Techniques: Western Blot, Immunofluorescence, Transfection

Journal: Precision Clinical Medicine

Article Title: Agrimol B inhibits pancreatic ductal adenocarcinoma by induction of lethal mitophagy through decreasing mitochondrial transcription termination factor 3

doi: 10.1093/pcmedi/pbag009

Figure Lengend Snippet: Agrimol B regulates mitophagy by downregulating MTERF3 expression. (A) Venn diagram showing the overlap of differentially expressed proteins (fold-change ≥ 1.3 or ≤ 0.76) between PANC-1 and AsPC-1 cells. (B, C) Volcano plots of DEGs identified via label-free quantitative proteomics in PANC-1 and AsPC-1 cells. (D) Western blot analysis of MTERF3 in PANC-1 and AsPC-1 cells treated with Agrimol B for 24 h. (E) Differences in MTERF3 expression between normal tissues and cancer tissues in the UCSC Xena database. (F) Kaplan-Meier analysis of MTERF3 expression and overall survival in 64 patients with PDAC. (G, H) CCK-8 assay in PDAC cells transfected with vector or oeMTERF3 following treatment with or without Agrimol B. (I) Western blot analysis of LC3 in PDAC cells transfected with vector or oeMTERF3 following treatment with or without Agrimol B. (J) Western blot analysis of PINK1 and Parkin in PDAC cells transfected with vector or oeMTERF3 following treatment with or without Agrimol B. (K) Immunohistochemical analyses of PINK1 and MTERF3 expression in PDAC tissues. Scale bars, 100 μm. (L) Correlation of the immunostaining intensities of PINK1 and MTERF3. (M) Western blot analysis of TIM23, SOD2, and HADHA in PDAC cells transfected with vector or oeMTERF3 following treatment with or without Agrimol B. (N) Molecular docking suggests that Agrimol B can bind to MTERF3 with a binding energy of -6.085 kcal/mol. (O) Western blot analysis of MTERF3 in cells treated with or without Agrimol B in the presence or absence of MG132.

Article Snippet: An anti-microtubule-associated protein light chain 3 (LC3) antibody (NB100-2220) was purchased from Novus, while anti-MTERF3 (EM1701-29) and lysosomal associated membrane protein 2 (LAMP2) (M1603-5) antibodies were purchased from HuaBio.

Techniques: Expressing, Quantitative Proteomics, Western Blot, CCK-8 Assay, Transfection, Plasmid Preparation, Immunohistochemical staining, Immunostaining, Binding Assay