Journal: iScience

Article Title: A calcium-sensing receptor dileucine motif directs internalization to spatially distinct endosomal signaling pathways

doi: 10.1016/j.isci.2025.112651

Figure Lengend Snippet: CaSR traffics to Rab4 pathways and recycles by Rab4 and Rab11 (A and B) BRET between Venus-Rab4 and CaSR-Nluc-WT or CaSR-Nluc-AA ( N = 7), or CaSR-Nluc-WT in HEK-AP2σ-WT or HEK-AP2σ-R15H cells ( N = 9), with (B) AUC. (C) BRET between Rab4-Venus and CaSR-Nluc-WT, with vehicle (DMSO) or Dyngo-4a. N = 5. (D) SIM of SNAP-CaSR-647 and Venus-Rab4. Scale, 5 μm. Arrows show colocalization. N = 23 (0 mM), N = 22 (3 mM) from N = 4 biological replicates. (E) SIM images of cells exposed to FLAG antibody and either non-permeabilized (total) or exposed to 0 or 3 mM Ca 2+ e for 30 min or 30 min with 3-h recovery, before acid strip to remove cell surface FLAG. Internalization occurs in cells stimulated with 3 mM Ca 2+ e conditions other than plates incubated at 4°C to block endocytosis. Cells transfected with Rab4 siRNA and Rab11-DN have reduced receptor recycling (seen as FLAG expression at cell surfaces) than cells transfected with scrambled siRNA or Rab11-WT. Scale, 5 μm. (F) Non-permeabilized cells incubated with FLAG antibody and exposed to 0 or 3 mM Ca 2+ e for 30 min or 30 min with 3-h recovery to monitor recycling. Comparisons to 3 mM (30 min) in black, to 3 mM (30 min with recovery) in red, and to 0 mM (30 min with recovery) in blue. N = 5. (G) Antibody feeding in cells transfected with scrambled siRNA, Rab4 siRNA, or Rab11a-dominant negative (DN). Comparisons to 0 min (blue). N = 5. (H) Antibody feeding assays in which permeabilized cells were incubated with FLAG antibody and exposed to 0 or 3 mM Ca 2+ e for 30 min or 30 min with 3-h recovery. Statistics show comparisons to 3 mM (30 min) in black, to 3 mM (30 min with recovery) in red, and to 0 mM (30 min with recovery) in blue. N = 5. (I) Antibody feeding assays in cells transfected with scrambled siRNA, Rab4 siRNA, or Rab11a-DN and permeabilized to monitor total CaSR. Statistical analyses show comparisons to 0 min in blue and between 0 and 3 mM in each condition in black. N = 4. Statistical analyses were performed using one-way ANOVA with Tukey’s multiple-comparisons test for (B), unpaired t test in (C), and one-way ANOVA with Sidak’s multiple-comparisons test in (F)–(I). ∗∗∗∗ p < 0.0001, ∗∗∗ p < 0.001, ∗∗ p < 0.01, ∗ p < 0.05. Data shows mean + SEM in A–C,and F–I.

Article Snippet: Rab4 (RAB4A) Human siRNA Oligo Duplex (Locus ID 5867) , Origene , Catalog. no. SR303946.

Techniques: Stripping Membranes, Incubation, Blocking Assay, Transfection, Expressing, Dominant Negative Mutation

Journal: iScience

Article Title: A calcium-sensing receptor dileucine motif directs internalization to spatially distinct endosomal signaling pathways

doi: 10.1016/j.isci.2025.112651

Figure Lengend Snippet: CaSR signaling from Rab4- and Rab9-positive endosomes contributes to sustained signaling (A) BRET between Venus-Rab4 and Nluc-mGq in HEK-CaSR. Calcium ( N = 9), spermine ( N = 6). (B) Ca 2+ -induced BRET with vehicle (DMSO) or Dyngo-4a. N = 8. (C and D) BRET between Venus-Rab4 and CaSR-Nluc-WT or CaSR-Nluc-AA or (D) CaSR-Nluc-WT in HEK-AP2σ-WT or HEK-AP2σ-R15H cells. N = 7. (E) AUC of ligand-induced BRET between Nluc-GRK3 and Venus-Gβγ in HEK-CaSR overexpressing Gα11, with scrambled or Rab4 siRNA. N = 8. (F) AUC of NanoBiT IP 3 responses with scrambled or Rab4 siRNA. N = 6. (G) Western blot of p -ERK1/2 in HEK-CaSR with scrambled or Rab4 siRNA following a 5-min pulse of 3 mM Ca 2+ then 0.1 mM Ca 2+ . Quantification of densitometry from five western blots is shown below. (H) BRET between Venus-Rab9 and Nluc-mGq in HEK-CaSR. Calcium ( N = 9), spermine ( N = 6). (I) BRET with vehicle (DMSO) or Dyngo-4a. N = 8. (J and K) Ca 2+ -induced BRET in (J) cells transfected with CaSR-Nluc-WT or CaSR-Nluc-AA ( N = 5) or in (K) HEK-AP2σ-WT or HEK-AP2σ-R15H cells ( N = 10). (L) BRET between Nluc-GRK3 and Venus-Gβγ in HEK-CaSR overexpressing Gα11, with scrambled or Rab9 siRNA. N = 8. (M) NanoBiT IP 3 responses with scrambled or Rab9 siRNA. N = 6. (N) p -ERK1/2 responses with scrambled or Rab4 siRNA and exposed to a 5-min pulse of 3 mM Ca 2+ e . Quantification of densitometry from four western blots is shown below. Data shows mean ± SEM. ∗∗∗∗ p < 0.0001, ∗∗∗ p < 0.001, ∗∗ p < 0.01, ∗ p < 0.05. Black asterisks show vehicle vs. agonist. Statistical analyses were performed using one-way ANOVA with Sidak’s multiple comparisons test for (A), (B), (C), (D), (G), (H), (I), (J), (K), and (N), and two-way ANOVA with Sidak’s test for (E), (F), (L), and (M).

Article Snippet: Rab4 (RAB4A) Human siRNA Oligo Duplex (Locus ID 5867) , Origene , Catalog. no. SR303946.

Techniques: Western Blot, Transfection

Journal: iScience

Article Title: A calcium-sensing receptor dileucine motif directs internalization to spatially distinct endosomal signaling pathways

doi: 10.1016/j.isci.2025.112651

Figure Lengend Snippet: Cinacalcet increases CaSR signaling in FHH3-associated AP2σ-R15H mutant cells by enhancing sustained signaling (A) BRET between Venus-Rab4 and CaSR-Nluc with vehicle or cinacalcet. (B and C) Dose response of BRET responses with increasing cinacalcet concentrations with (C) pEC50 values. N = 4. (D and E) BRET between Venus-Rab4 and CaSR-Nluc with vehicle or NPS-2143, with (E) dose response. N = 6. (F and G) BRET between Venus-Rab4 and Nluc-mGq with increasing cinacalcet concentrations, with (G) pEC50. N = 4. (H) BRET between Nluc-mGq and Venus-Rab4 with NPS-2143. N = 12. (I–K) BRET between Nluc-mGq and Venus-Rab9 with cinacalcet, with (J) increasing cinacalcet concentrations, and (K) pEC50. N = 5. (L and M) BRET between Nluc-mGq and Venus-Rab9 in cells with increasing concentrations of cinacalcet, with (M) pEC50. N = 5. (N) BRET between Nluc-CaSR and Venus-Rab9 with NPS-2143. N = 12. (O) BRET between Nluc-mGq and Venus-Rab9 with NPS-2143. N = 11. (P–R) BRET between Nluc-mGq and (P) Rab5 ( N = 6), (Q) Rab4 ( N = 4), (R) Rab9 ( N = 5), in AP2σ-WT or AP2σ-R15H cells expressing CaSR with vehicle or cinacalcet. (S and T) Cinacalcet effect on responses in cells expressing a siRNA-resistant AP2σ-R15H plasmid combined with AP2σ-siRNA to knockdown endogenous protein or an AP2σ-R15H plasmid. N = 5. Statistics show: WT vs. AP2σ-R15H (red), AP2σ-R15H with vehicle or cinacalcet (blue) in (P)–(R). Statistical analyses were performed using two-way ANOVA with Dunnett’s test in (E), and Sidak’s test in (H,) (N), (O), (P), (Q), (R), (S), and (T), and one-way ANOVA with Dunnett’s test in (C), (G), (K), and (M). ∗∗∗∗ p < 0.0001, ∗∗∗ p < 0.001, ∗∗ p < 0.01, ∗ p < 0.05. (A), (D), and (I) show examples of BRET data. Data shows mean ± SEM.

Article Snippet: Rab4 (RAB4A) Human siRNA Oligo Duplex (Locus ID 5867) , Origene , Catalog. no. SR303946.

Techniques: Mutagenesis, Expressing, Plasmid Preparation, Knockdown

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