Journal: Frontiers in Immunology

Article Title: Chlamydia trachomatis Infection Impairs MHC-I Intracellular Trafficking and Antigen Cross-Presentation by Dendritic Cells

doi: 10.3389/fimmu.2021.662096

Figure Lengend Snippet: C. trachomatis recruits Rab14, Rab4, Rab22a, and Rab11a to the inclusion in JAWS-II DCs. JAWS-II DCs were infected with GFP- C. trachomatis L2 at MOI 100 for 24 h and analyzed by confocal microscopy. The intracellular distribution of (A, B) Rab14, (E, F) Rab4, (I, J) Rab22a and (M, N) Rab11a was evaluated in non-infected and infected cells. Endogenous Rab proteins were detected by indirect immunofluorescence using primary antibodies followed by its corresponding Cy3-conjugated secondary antibodies. Insets show a chlamydial inclusion magnification for each marker. DAPI was used to stain DNA. Bars represent 10 um. Images are representative of three independent experiments and more than 20 images were analyzed for each Rab. (C, G, K, O) A representative chlamydial inclusion was transversely crossed with eight diameter lines to obtain an intensity histogram. Line graphs show the MFI from GFP and the corresponding Rab protein with the SD for each point. (D, H, L, P) Chlamydial inclusion was transversely crossed with a diameter line to obtain an intensity histogram for each Rab protein; and five points of each line corresponding to the cytoplasm (P1), inclusion membrane (P2), inside the inclusion (P3), inclusion membrane (P4), and cytoplasm (P5) were selected. Twenty cells were analyzed for each Rab protein. One-way ANOVA with Dunnett’s multiple comparison post-test were performed. ### P < 0.001, #### P < 0.0001, and ****P < 0.0001.

Article Snippet: The following primaries antibodies were used: rabbit anti-CT529 (gently provided by Agathe Subtil, Pasteur Institute, Paris, France), rabbit polyclonal anti-Rab14, Rab4 and Rab11a (Aviva Systems Biology), mouse anti-CD63 (Invitrogen), goat polyclonal anti-EEA1 (Santa Cruz), purified mouse anti-Lamp1, purified mouse anti-H-2K b and FITC-coupled mouse anti-H-2K b (BD Biosciences), mouse monoclonal anti-TfR H68.4 (Invitrogen), mouse monoclonal anti-Rab22a (Santa Cruz), rabbit anti-clathrin (Abcam), rabbit anti-OVA (Sigma-Aldrich), and rabbit anti-MOMP (generously provided by Ted Hackstadt, National Institutes of Health, USA).

Techniques: Infection, Confocal Microscopy, Immunofluorescence, Marker, Staining, Membrane, Comparison

Journal: Cancer Science

Article Title: Podocalyxin‐like protein promotes gastric cancer progression through interacting with RUN and FYVE domain containing 1 protein

doi: 10.1111/cas.13864

Figure Lengend Snippet: Immunohistochemical analysis of podocalyxin‐like protein (PODXL) and RUN and FYVE domain containing 1 (RUFY1) expression in gastric cancer (GC) tissues and the association of expression levels with the prognosis of GC patients. A, Representative images of PODXL and RUFY1 staining in GC tissues and their corresponding normal‐appearing tissues (NAT) (400×). B, C, Relative percentage of PODXL and RUFY1 positive staining in GC tissues and their corresponding NAT. D, A significant positive correlation between PODXL and RUFY1 expression was detected. E, F, Survival analysis showed that GC patients with high tissue levels of PODX or RUFY1 had a worse 5‐year OS compared with patients with low levels of each protein (* P < .05)

Article Snippet: Antibodies against PODXL (#150358 for western blotting and #205350 for co‐immunoprecipitation [co‐IP]) and RUFY1 (13498‐1‐AP) were purchased from Abcam and Proteintech (USA), respectively.

Techniques: Immunohistochemical staining, Expressing, Staining

Journal: Cancer Science

Article Title: Podocalyxin‐like protein promotes gastric cancer progression through interacting with RUN and FYVE domain containing 1 protein

doi: 10.1111/cas.13864

Figure Lengend Snippet: Relationships between PODXL (or RUFY1) and clinicopathological factors in 130 gastric cancer tissues

Article Snippet: Antibodies against PODXL (#150358 for western blotting and #205350 for co‐immunoprecipitation [co‐IP]) and RUFY1 (13498‐1‐AP) were purchased from Abcam and Proteintech (USA), respectively.

Techniques:

Journal: Cancer Science

Article Title: Podocalyxin‐like protein promotes gastric cancer progression through interacting with RUN and FYVE domain containing 1 protein

doi: 10.1111/cas.13864

Figure Lengend Snippet: Analysis of the expression of podocalyxin‐like protein (PODXL) and RUN and FYVE domain containing 1 (RUFY1) mRNA in gastric cancer (GC) serum and the association of expression levels with the prognosis of GC patients. A, Serum PODXL levels in GC patients were significantly increased compared with those of healthy volunteers (normal group). B‐D, Serum PODXL levels in GC patients with positive lymph node metastases (Lx group), advanced stage (III‐IV) or distant metastases (M1) were higher than those without lymph node metastases (L0), of early stage (I‐II) or without distant metastases (M0). E, Serum RUFY1 levels in GC patients were significantly increased compared with those of healthy volunteers (normal group). F‐H, Serum RUFY1 levels in GC patients with positive lymph node metastases (Lx group), advanced stage (III‐IV stage) or distant metastases (M1) were higher than those without lymph node metastases (L0), of early stage (I‐II stage) or without distant metastases (M0). I, PODXL mRNA levels in the serum of GC patients were significantly associated with RUFY1 ( r = .596 P = .000). J and K, Survival analysis indicated that GC patients with high levels of serum PODXL or RUFY1 had a shorter OS compared with those with low PODXL or RUFY1 levels (* P < .05)

Article Snippet: Antibodies against PODXL (#150358 for western blotting and #205350 for co‐immunoprecipitation [co‐IP]) and RUFY1 (13498‐1‐AP) were purchased from Abcam and Proteintech (USA), respectively.

Techniques: Expressing

Journal: Cancer Science

Article Title: Podocalyxin‐like protein promotes gastric cancer progression through interacting with RUN and FYVE domain containing 1 protein

doi: 10.1111/cas.13864

Figure Lengend Snippet: Podocalyxin‐like protein (PODXL) interacted with RUN and FYVE domain containing 1 (RUFY1) in gastric cancer (GC) cells in vitro. A, B, Mass spectrometry identified MFF, GRP78, TGM3, RUFY1, FSH6Q2, CALL5, TBA1B and ACTB as possible interacted partners of PODXL. C, Co‐immunoprecipitation assays detected PODXL/RUFY1 complexes in SGC‐7901 cells. D, Western blotting revealed that re‐expressing PODXL significantly increased the levels of RUFY1, while knocking down RUFY1 expression did not change the PODXL expression in siRNA‐RUFY1 or PODXL‐siRNA‐RUFY1 cells (* P < .05)

Article Snippet: Antibodies against PODXL (#150358 for western blotting and #205350 for co‐immunoprecipitation [co‐IP]) and RUFY1 (13498‐1‐AP) were purchased from Abcam and Proteintech (USA), respectively.

Techniques: In Vitro, Mass Spectrometry, Immunoprecipitation, Western Blot, Expressing

Journal: Cancer Science

Article Title: Podocalyxin‐like protein promotes gastric cancer progression through interacting with RUN and FYVE domain containing 1 protein

doi: 10.1111/cas.13864

Figure Lengend Snippet: RUN and FYVE domain containing 1 (RUFY1) knockdown attenuated podocalyxin‐like protein (PODXL)‐induced biological behaviors in vitro. A, The CCK‐8 assay revealed that RUFY1 knockdown significantly inhibited the proliferation of gastric cancer (GC) cells at 48, 72 and 96 h and attenuated the proliferative effects of PODXL overexpression at 48, 72 and 96 h. B, Western blotting showed that RUFY1 knockdown altered the levels of caspase‐3, Bax, Bcl‐2, MMP‐2 and MMP9, and attenuated the PODXL‐induced changes of these protein expressions. C and D, RUFY1 knockdown inhibited the colony formation and attenuated PODXL‐induced colony formation. E, F, Hoechst33342 staining showed that RUFY1 knockdown increased apoptosis and attenuated the PODXL‐induced changes in apoptosis (* P < .05)

Article Snippet: Antibodies against PODXL (#150358 for western blotting and #205350 for co‐immunoprecipitation [co‐IP]) and RUFY1 (13498‐1‐AP) were purchased from Abcam and Proteintech (USA), respectively.

Techniques: Knockdown, In Vitro, CCK-8 Assay, Over Expression, Western Blot, Staining

Journal: Cancer Science

Article Title: Podocalyxin‐like protein promotes gastric cancer progression through interacting with RUN and FYVE domain containing 1 protein

doi: 10.1111/cas.13864

Figure Lengend Snippet: RUN and FYVE domain containing 1 (RUFY1) knockdown attenuated podocalyxin‐like protein (PODXL)‐induced biological behaviors and inactivated the PI3K/AKT, NF‐κB and MAPK/ERK signaling pathways in vitro. A, B, RUFY1 knockdown significantly inhibited the wound healing and attenuated the PODXL‐induced changes in wound healing. C‐F, RUFY1 knockdown significantly inhibited migration and invasion and attenuated the PODXL‐induced changes in migration and invasion. G, H, RUFY1 knockdown significantly inhibited the phosphorylation of PI3K, AKT, MAPK, NF‐κB and ERK1 without significantly changing the basal level of protein expression and attenuated the PODXL‐induced changes in the activation of signaling pathways (* P < .05)

Article Snippet: Antibodies against PODXL (#150358 for western blotting and #205350 for co‐immunoprecipitation [co‐IP]) and RUFY1 (13498‐1‐AP) were purchased from Abcam and Proteintech (USA), respectively.

Techniques: Knockdown, Protein-Protein interactions, In Vitro, Migration, Phospho-proteomics, Expressing, Activation Assay

Journal: Cancer Science

Article Title: Podocalyxin‐like protein promotes gastric cancer progression through interacting with RUN and FYVE domain containing 1 protein

doi: 10.1111/cas.13864

Figure Lengend Snippet: RUN and FYVE domain containing 1 (RUFY1) knockdown attenuated podocalyxin‐like protein (PODXL)‐induced tumorigenesis in vivo. A, RUFY1 knockdown significantly inhibited tumorigenesis in nude mice and attenuated the PODXL‐induced changes in tumor formation. B, Tumor growth curves were generated each week. C, Tumor weights were compared. D, E, Histological analysis revealed that RUFY1 knockdown significantly decreased the expression of Ki67 and the levels of p‐Akt and p‐NF‐κB expressions, and attenuated the PODXL‐induced changes of signaling proteins (* P < .05)

Article Snippet: Antibodies against PODXL (#150358 for western blotting and #205350 for co‐immunoprecipitation [co‐IP]) and RUFY1 (13498‐1‐AP) were purchased from Abcam and Proteintech (USA), respectively.

Techniques: Knockdown, In Vivo, Generated, Expressing

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: Developmental cell

Article Title: Membrane flow drives an adhesion-independent amoeboid cell migration mode

doi: 10.1016/j.devcel.2018.05.029

Figure Lengend Snippet: Data and Software Availability

Article Snippet: The following constructs were obtained from Addgene: mVenus-myosinIIA (#56389, Michael Davidson), GFP-rGBD (#26732, William Bement), mCherry-ezrin (#55043, Michael Davidson), mCherry-moesin (#55103, Michael Davidson), Venus-iLID-CaaX (#60411, Brian Kuhlman), mRFP-FKBP12-5ptpase (#67516, Tamas Balla), mRFP-FKBP12 (#67514, Tamas Balla), EYFP-Clathrin-19 (#56584, Michael Davidson), mCherry-Lifeact-7 (#54491, Michael Davidson), mApple-caveolin (#54872, Michael Davidson), pUASP-YFP-Rab4 (#37690, Matthew Scott), pUASP-YFP-Rab4DN (#37691, Matthew Scott).

Techniques: Software, Recombinant, Sequencing, RNA Sequencing Assay