Journal: Frontiers in Pharmacology

Article Title: Loss of SynDIG4/PRRT1 alters distribution of AMPA receptors in Rab4- and Rab11-positive endosomes and impairs basal AMPA receptor recycling

doi: 10.3389/fphar.2025.1568908

Figure Lengend Snippet: Accumulation of AMPARs in Rab4-positive endosomes in SynDIG4 KO neurons (A) Representative images showing the colocalization of EEA1 and GluA1 and quantification of Manders’ colocalization coefficient for EEA1 and GluA1 in WT and SynDIG4 KO neurons. (B) Representative images showing the colocalization of Rab11 and GluA1 and quantification of Manders’ colocalization coefficient for Rab11 and GluA1 in WT and SynDIG4 KO neurons. (C) Representative images showing the colocalization of Rab4 and GluA1 and quantification of Manders’ colocalization coefficient for Rab4 and GluA1 in WT and SynDIG4 KO neurons. (D) Representative images showing the colocalization of EEA1 and GluA2 and quantification of Manders’ colocalization coefficient for EEA1 and GluA2 in WT and SynDIG4 KO neurons. (E) Representative images showing the colocalization of Rab11 and GluA2 and quantification of Manders’ colocalization coefficient for Rab11 and GluA2 in WT and SynDIG4 KO neurons. (F) Representative images showing the colocalization of Rab4 and GluA2 and quantification of Manders’ colocalization coefficient for Rab4 and GluA2 in WT and SynDIG4 KO neurons. Data are presented as mean ± SEM, with statistical significance determined using unpaired t-test; n = ∼40 dendrite stretches, 2–3 dendritic stretches were cropped from individual neuron images; **p < 0.01, ***p < 0.001, ns, not significant. Three biological replicates (independent cultures, each from a different litter with paired WT and KO littermates) were performed for each group, and representative results shown are from one of these replicates. Scale bar, 5 µm.

Article Snippet: The primary antibodies included MAP2 (Millipore, Cat# AB5622-1, 1:300), PSD95 (Synaptic Systems, Cat# 124014, 1:300), GluA1-C (Millipore, Cat# AB1504, 1:200), GluA2-C (NeuroMab, SKU: 75-002, 1:200), EEA1 (Cell Signaling, Cat# 2411, 1:100), Rab4 (Cell Signaling, Cat# 2167, 1:50), Rab4 (Santa Cruz, Cat# sc-517263, 1:50), and Rab11 (Cell Signaling, Cat# 5589, 1:50), and Rab11 (ThermoFisher, Cat# MA5-49197, 1:50).

Techniques:

Journal: Frontiers in Pharmacology

Article Title: Loss of SynDIG4/PRRT1 alters distribution of AMPA receptors in Rab4- and Rab11-positive endosomes and impairs basal AMPA receptor recycling

doi: 10.3389/fphar.2025.1568908

Figure Lengend Snippet: Increased colocalization of Rab4-positive and Rab11-positive compartments in SynDIG4 KO neurons (A) Representative images showing the colocalization of SynDIG4 and Rab4 or Rab11. (B) Representative images showing the colocalization of Rab4 and Rab11. (C) Representative images showing the colocalization of Rab4 and EEA1. (D) Quantification of Manders’ colocalization coefficient for SynDIG4 and Rab4 or Rab11 in WT neurons. (E) Quantification of Manders’ colocalization coefficient for Rab4 and Rab11 in WT and SynDIG4 KO neurons. (F) Quantification of Manders’ colocalization coefficient for Rab4 and EEA1 in WT and SynDIG4 KO neurons. (G–I) Quantification of puncta size of Rab4, Rab11 and EEA1 in WT and SynDIG4 KO neurons, respectively. Data are presented as mean ± SEM, with statistical significance determined using unpaired t-test; n = ∼40 dendrite stretches, 2–3 dendritic stretches were cropped from individual neuron images; ****p < 0.0001, ns, not significant. Three biological replicates (independent cultures, each from a different litter with paired WT and KO littermates) were performed for each group, and representative results shown are from one of these replicates. Scale bar, 5 µm.

Article Snippet: The primary antibodies included MAP2 (Millipore, Cat# AB5622-1, 1:300), PSD95 (Synaptic Systems, Cat# 124014, 1:300), GluA1-C (Millipore, Cat# AB1504, 1:200), GluA2-C (NeuroMab, SKU: 75-002, 1:200), EEA1 (Cell Signaling, Cat# 2411, 1:100), Rab4 (Cell Signaling, Cat# 2167, 1:50), Rab4 (Santa Cruz, Cat# sc-517263, 1:50), and Rab11 (Cell Signaling, Cat# 5589, 1:50), and Rab11 (ThermoFisher, Cat# MA5-49197, 1:50).

Techniques:

Journal: Frontiers in Pharmacology

Article Title: Loss of SynDIG4/PRRT1 alters distribution of AMPA receptors in Rab4- and Rab11-positive endosomes and impairs basal AMPA receptor recycling

doi: 10.3389/fphar.2025.1568908

Figure Lengend Snippet: Model for the role of SynDIG4 in AMPAR distribution via endosomal recycling mechanism. In WT neurons, SynDIG4 (SD4) facilitates the trafficking of AMPARs between Rab4-positive and Rab11-positive endosomes under baseline conditions. In SynDIG4 KO neurons, the recycling of GluA1-containing AMPARs is reduced, and Rab11-positive endosomes fail to undergo fission from Rab4-positive endosomes. Consequently, the levels of intracellular AMPARs are increased, impairing the Rab4-Rab11-dependent recycling process. This SD4-mediated mechanism is crucial for the synaptic distribution of surface AMPARs. Created in BioRender. He, C. (2025) https://BioRender.com/k52o982 .

Article Snippet: The primary antibodies included MAP2 (Millipore, Cat# AB5622-1, 1:300), PSD95 (Synaptic Systems, Cat# 124014, 1:300), GluA1-C (Millipore, Cat# AB1504, 1:200), GluA2-C (NeuroMab, SKU: 75-002, 1:200), EEA1 (Cell Signaling, Cat# 2411, 1:100), Rab4 (Cell Signaling, Cat# 2167, 1:50), Rab4 (Santa Cruz, Cat# sc-517263, 1:50), and Rab11 (Cell Signaling, Cat# 5589, 1:50), and Rab11 (ThermoFisher, Cat# MA5-49197, 1:50).

Techniques:

Journal: Frontiers in Pharmacology

Article Title: Loss of SynDIG4/PRRT1 alters distribution of AMPA receptors in Rab4- and Rab11-positive endosomes and impairs basal AMPA receptor recycling

doi: 10.3389/fphar.2025.1568908

Figure Lengend Snippet: Accumulation of AMPARs in Rab4-positive endosomes in SynDIG4 KO neurons (A) Representative images showing the colocalization of EEA1 and GluA1 and quantification of Manders’ colocalization coefficient for EEA1 and GluA1 in WT and SynDIG4 KO neurons. (B) Representative images showing the colocalization of Rab11 and GluA1 and quantification of Manders’ colocalization coefficient for Rab11 and GluA1 in WT and SynDIG4 KO neurons. (C) Representative images showing the colocalization of Rab4 and GluA1 and quantification of Manders’ colocalization coefficient for Rab4 and GluA1 in WT and SynDIG4 KO neurons. (D) Representative images showing the colocalization of EEA1 and GluA2 and quantification of Manders’ colocalization coefficient for EEA1 and GluA2 in WT and SynDIG4 KO neurons. (E) Representative images showing the colocalization of Rab11 and GluA2 and quantification of Manders’ colocalization coefficient for Rab11 and GluA2 in WT and SynDIG4 KO neurons. (F) Representative images showing the colocalization of Rab4 and GluA2 and quantification of Manders’ colocalization coefficient for Rab4 and GluA2 in WT and SynDIG4 KO neurons. Data are presented as mean ± SEM, with statistical significance determined using unpaired t-test; n = ∼40 dendrite stretches, 2–3 dendritic stretches were cropped from individual neuron images; **p < 0.01, ***p < 0.001, ns, not significant. Three biological replicates (independent cultures, each from a different litter with paired WT and KO littermates) were performed for each group, and representative results shown are from one of these replicates. Scale bar, 5 µm.

Article Snippet: The primary antibodies included MAP2 (Millipore, Cat# AB5622-1, 1:300), PSD95 (Synaptic Systems, Cat# 124014, 1:300), GluA1-C (Millipore, Cat# AB1504, 1:200), GluA2-C (NeuroMab, SKU: 75-002, 1:200), EEA1 (Cell Signaling, Cat# 2411, 1:100), Rab4 (Cell Signaling, Cat# 2167, 1:50), Rab4 (Santa Cruz, Cat# sc-517263, 1:50), and Rab11 (Cell Signaling, Cat# 5589, 1:50), and Rab11 (ThermoFisher, Cat# MA5-49197, 1:50).

Techniques:

Journal: Frontiers in Pharmacology

Article Title: Loss of SynDIG4/PRRT1 alters distribution of AMPA receptors in Rab4- and Rab11-positive endosomes and impairs basal AMPA receptor recycling

doi: 10.3389/fphar.2025.1568908

Figure Lengend Snippet: Increased colocalization of Rab4-positive and Rab11-positive compartments in SynDIG4 KO neurons (A) Representative images showing the colocalization of SynDIG4 and Rab4 or Rab11. (B) Representative images showing the colocalization of Rab4 and Rab11. (C) Representative images showing the colocalization of Rab4 and EEA1. (D) Quantification of Manders’ colocalization coefficient for SynDIG4 and Rab4 or Rab11 in WT neurons. (E) Quantification of Manders’ colocalization coefficient for Rab4 and Rab11 in WT and SynDIG4 KO neurons. (F) Quantification of Manders’ colocalization coefficient for Rab4 and EEA1 in WT and SynDIG4 KO neurons. (G–I) Quantification of puncta size of Rab4, Rab11 and EEA1 in WT and SynDIG4 KO neurons, respectively. Data are presented as mean ± SEM, with statistical significance determined using unpaired t-test; n = ∼40 dendrite stretches, 2–3 dendritic stretches were cropped from individual neuron images; ****p < 0.0001, ns, not significant. Three biological replicates (independent cultures, each from a different litter with paired WT and KO littermates) were performed for each group, and representative results shown are from one of these replicates. Scale bar, 5 µm.

Article Snippet: The primary antibodies included MAP2 (Millipore, Cat# AB5622-1, 1:300), PSD95 (Synaptic Systems, Cat# 124014, 1:300), GluA1-C (Millipore, Cat# AB1504, 1:200), GluA2-C (NeuroMab, SKU: 75-002, 1:200), EEA1 (Cell Signaling, Cat# 2411, 1:100), Rab4 (Cell Signaling, Cat# 2167, 1:50), Rab4 (Santa Cruz, Cat# sc-517263, 1:50), and Rab11 (Cell Signaling, Cat# 5589, 1:50), and Rab11 (ThermoFisher, Cat# MA5-49197, 1:50).

Techniques:

Journal: Frontiers in Pharmacology

Article Title: Loss of SynDIG4/PRRT1 alters distribution of AMPA receptors in Rab4- and Rab11-positive endosomes and impairs basal AMPA receptor recycling

doi: 10.3389/fphar.2025.1568908

Figure Lengend Snippet: Model for the role of SynDIG4 in AMPAR distribution via endosomal recycling mechanism. In WT neurons, SynDIG4 (SD4) facilitates the trafficking of AMPARs between Rab4-positive and Rab11-positive endosomes under baseline conditions. In SynDIG4 KO neurons, the recycling of GluA1-containing AMPARs is reduced, and Rab11-positive endosomes fail to undergo fission from Rab4-positive endosomes. Consequently, the levels of intracellular AMPARs are increased, impairing the Rab4-Rab11-dependent recycling process. This SD4-mediated mechanism is crucial for the synaptic distribution of surface AMPARs. Created in BioRender. He, C. (2025) https://BioRender.com/k52o982 .

Article Snippet: The primary antibodies included MAP2 (Millipore, Cat# AB5622-1, 1:300), PSD95 (Synaptic Systems, Cat# 124014, 1:300), GluA1-C (Millipore, Cat# AB1504, 1:200), GluA2-C (NeuroMab, SKU: 75-002, 1:200), EEA1 (Cell Signaling, Cat# 2411, 1:100), Rab4 (Cell Signaling, Cat# 2167, 1:50), Rab4 (Santa Cruz, Cat# sc-517263, 1:50), and Rab11 (Cell Signaling, Cat# 5589, 1:50), and Rab11 (ThermoFisher, Cat# MA5-49197, 1:50).

Techniques:

Journal: Biology direct

Article Title: Nucleolin in the cell membrane promotes Ang II-mediated VSMC phenotypic switching by regulating the AT1R internalization function : Nucleolin promotes Ang II-mediated VSMC phenotypic switching.

doi: 10.1186/s13062-025-00615-0

Figure Lengend Snippet: Fig. 5 NCL delays Ang II-induced AT1R internalization and recruits Rab4 and Rab11 to promote recycling by inhibiting AT1R phosphorylation. A. RT-qPCR and WB analysis of NCL expression in VSMCs transfected with oe-NC or oe-NCL. *p < 0.05 vs. oe-NC. B. WB analysis of AT1R expression in the cell mem brane and cytoplasm of VSMCs. C. WB analysis of β-arrestin2, Clathrin, AP-2, and Caveolae-1 expression in VSMCs. D. Detection of AT1R phosphorylation in VSMCs. Pulldown Westerns are indicated by pulldown (PD). E. Co-IP assay for validating the binding of AT1R to Rab4 and Rab11, respectively. F. WB analysis of Rab4 and Rab11expression in VSMCs. VSMCs were transfected with oe-NC or oe-NCL and then treated with Ang II. *p < 0.05 vs. Control, #p < 0.05 vs. Ang II + oe-NC. n = 3

Article Snippet: To detect the interaction of AT1R with Rab4, and Rab11, proteins were incubated with normal rabbit IgG (B900610, Proteintech) or AT1R antibody (25343-1-AP, Proteintech).

Techniques: Phospho-proteomics, Quantitative RT-PCR, Expressing, Transfection, Co-Immunoprecipitation Assay, Binding Assay, Control

Journal: Biology direct

Article Title: Nucleolin in the cell membrane promotes Ang II-mediated VSMC phenotypic switching by regulating the AT1R internalization function : Nucleolin promotes Ang II-mediated VSMC phenotypic switching.

doi: 10.1186/s13062-025-00615-0

Figure Lengend Snippet: Fig. 6 NCL induces VSMC phenotypic switching by reducing Ang II-induced AT1R internalization leading to sustained AT1R activation. VSMCs were transfected with oe-NC or oe-NCL and then treated with Ang II and 50 μm rasarfin. A. WB analysis of β-arrestin2, Clathrin, AP-2, and Caveolae-1 expression in VSMCs. B. Detection of AT1R phosphorylation in VSMCs. C. WB analysis of Rab4 and Rab11expression in VSMCs. D. EDU assay for detecting cell prolifera tion. Scale bar = 50 µM. E. Scratch assay for detecting cell migration. Scale bar = 100 µM. F. WB analysis of α-SMA, SM22α, and OPN expression. G. IF assay for detecting α-SMA and OPN expression. Scale bar = 25 µM. VSMCs were transfected with sh-NC or sh-NCL and then treated with Ang II and rasarfin. *p < 0.05 vs. Ang II + sh-NC, #p < 0.05 vs. Ang II + sh-NCL + DMSO. n = 3

Article Snippet: To detect the interaction of AT1R with Rab4, and Rab11, proteins were incubated with normal rabbit IgG (B900610, Proteintech) or AT1R antibody (25343-1-AP, Proteintech).

Techniques: Activation Assay, Transfection, Expressing, Phospho-proteomics, EdU Assay, Wound Healing Assay, Migration

Journal: Biology direct

Article Title: Nucleolin in the cell membrane promotes Ang II-mediated VSMC phenotypic switching by regulating the AT1R internalization function : Nucleolin promotes Ang II-mediated VSMC phenotypic switching.

doi: 10.1186/s13062-025-00615-0

Figure Lengend Snippet: Fig. 7 NCL promotes VSMC phenotypic switching in mice. A. RT-qPCR and WB analysis of NCL expression in mice. B. Blood pressure monitoring in mice. C. H&E staining of thoracic aorta from mice. Scale bar = 100 and 25 µM. D. WB analysis of NCL expression. E. IF staining for detecting Ki67 expression. Scale bar = 25 µM. F. WB analysis of β-arrestin1, β-arrestin2, Clathrin, AP-2, and Caveolae-1 expression. G. WB analysis of Rab4 and Rab11expression. H. IF assay for detecting α-SMA and OPN expression. Scale bar = 25 µM. I. WB analysis of AT1R, p-p38MAPK/p38MAPK, p-JNK/JNK, p-ERK/ERK, and p-PKCα/ PKCα expression. The mice were treated with Ang II and injected with sh-NC or sh-NCL lentiviruses in the tail vein. *p < 0.05 vs. sh-NC. *p < 0.05 vs. Sham, #p < 0.05 vs. Ang II + sh-NC. n = 5

Article Snippet: To detect the interaction of AT1R with Rab4, and Rab11, proteins were incubated with normal rabbit IgG (B900610, Proteintech) or AT1R antibody (25343-1-AP, Proteintech).

Techniques: Quantitative RT-PCR, Expressing, Staining, Injection

Journal: The EMBO Journal

Article Title: Modulation of tumor inflammatory signaling and drug sensitivity by CMTM4

doi: 10.1038/s44318-024-00330-y

Figure Lengend Snippet: Reagents and tools table

Article Snippet: Rab4 , Cell Signaling Technology , 2167S.

Techniques: Recombinant, Western Blot, Immunohistochemistry, Plasmid Preparation, Sequencing, shRNA, Knock-Out, Isolation, Magnetic Beads, Luciferase, Reverse Transcription, SYBR Green Assay, Enzyme-linked Immunosorbent Assay, Software, Microscopy