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: The EMBO Journal

Article Title: TDP‐43 loss of function inhibits endosomal trafficking and alters trophic signaling in neurons

doi: 10.15252/embj.201694221

Figure Lengend Snippet: Primary hippocampal neurons (DIV6+3) were transfected with either shTDP or shCtrl together with GFP‐tagged organelle markers. A–C Mito‐GFP visualizes trafficking of mitochondria. At least four dendrite segments per neuron were live‐imaged with 2 Hz for 2.5 min. Quantitative analysis of relative vesicle movement (B) and vesicle number (C) from kymographs. D–F GFP‐RAB5 visualizes early endosomes. At least four dendrite segments per neuron were live‐imaged with 2 Hz for 2.5 min. Quantitative analysis of relative vesicle movement (E) and vesicle number (F) from kymographs. G–I GFP‐RAB4 visualizes fast recycling endosomes. At least four dendrite segments per neuron were live‐imaged with 2 Hz for 2.5 min. Quantitative analysis of relative vesicle movement (H) and vesicle number (I) from kymographs. Data information: Mean ± s.e.m., n = 3, unpaired, two‐tailed t ‐test: no significant differences were detected.

Article Snippet: Human RAB4 (Addgene #54943, M. Davidson unpublished), RAB5 (Lang et al , ), RAB7 and RAB11a wild type and S25N (Addgene #12605, 12674, 12678, Richard Pagano (Choudhury et al , )) were expressed from pEGFP‐C1 vector.

Techniques: Transfection, Two Tailed Test