Journal: American Journal of Physiology - Renal Physiology

Article Title: ILK and cytoskeletal architecture: an important determinant of AQP2 recycling and subsequent entry into the exocytotic pathway

doi: 10.1152/ajprenal.00336.2016

Figure Lengend Snippet: Immunofluorescence staining showing that Rab11 also accumulates in the perinuclear region after ILK inhibition. Control cells stained with Rab11 or AQP2 or both show a baseline scattered distribution of both AQP2 and Rab11, while those treated with cpd22 or ILK siRNA show a colocalization of both AQP2 and Rab11 in the perinuclear region. Scale, 20 μm. Green, AQP2; red, Rab11.

Article Snippet: Rabbit polyclonal anti- Rab11 antibody (1:200) was from BD Biosciences (San Jose, CA).

Techniques: Immunofluorescence, Staining, Inhibition

Journal: Traffic (Copenhagen, Denmark)

Article Title: Distinct motifs of neuropeptide Y receptors differentially regulate trafficking and desensitization.

doi: 10.1111/j.1600-0854.2007.00691.x

Figure Lengend Snippet: Figure 7: Colocalization of NPY activated EGFP-tagged Y1 receptors with transferrin–Texas Red, mCherry-tagged Rab5a and endogenous Rab4 and Rab11. HEK293 cells stably expressing EGFP Y1 receptors were coincubated with transferrin–Texas Red (5 mg/mL) and 100 nM NPY for 30 min before fixation and visualization by confocal microscopy. EGFP-Y1-expressing cells were transiently transfected with mCherry–Rab5a and activated with 100 nM NPY for 30 min before fixation and visualization by confocal microscopy. For Rab4 and Rab11 localization, the same activation protocol was used on EGFP Y1-expressing cells, but after fixation, cells were permeabilized and immunostained as described in Materials and Methods. Colocalized EGFP signals with transferrin–Texas Red, mCherry– Rab5a, Rab4 or Rab11 appear yellow in the merge picture. White boxes identify selected areas for which an enlarged section is shown. Individual confocal sections are shown. Scale bar represents 10 mm.

Article Snippet: Staining with primary antibodies (1/100 dilution) was of 40 min in PBS–1% BSA–0.5% saponin with purified mouse anti-Rab4 monoclonal antibody (BD Bioscience) or rabbit polyclonal IgG anti-Rab11 antibody (Santa Cruz).

Techniques: Stable Transfection, Expressing, Confocal Microscopy, Transfection, Activation Assay

Journal: bioRxiv

Article Title: The endocytic recycling pathway is controlled by the ADP-ribosylated GTPase Rab14

doi: 10.1101/2022.11.26.517555

Figure Lengend Snippet: Schematic representation of MARylated Rab14 role during endosomal progression. GTP-loaded Rab14 binds the effector RUFY1 on early/sorting endosomes. Endocytic stimuli, such as transferrin bound to its cognate receptor, cause PARP12 translocation to early/sorting endosomes, where Rab14 MARylation occurs. The ADP-ribose likely induces Rab14 conformational changes suitable for binding to the downstream effectors FIP1c/RCP (FIP1C) and Rab11, thus allowing endosome progression towards the endocytic recycling compartment (ERC). Here, GDP-bound Rab14 is released from endosomes, while GTP-loaded Rab11/FIP1C complex allows recycling of endosomes to the plasma membrane. At the ERC, an ADP-ribosylhydrolase (ADPrH) may remove the ADP-ribose moiety, making Rab14 available for the following cycle.

Article Snippet: Commercially available antibodies: mouse monoclonal antibodies to Transferrin Receptor/CD71 (clone H68.4; #13-6890, Thermo Fisher Scientific; western blots (WB) 1:3,000; immunofluorescence (IF) 1:200), EEA1 (#610456, BD Transduction Laboratories; WB 1:1,000; IF 1:100), Golgin-97 (CDF4, #A-21270, Thermo Fisher Scientific; IF 1:100); rabbit polyclonal antibodies to Rab4 (#ab13252, Sigma-Aldrich; IF 1:100), Rab14 (#R0656; Sigma-Aldrich, WB 1:2,000, IF 1:100), Rab11 (#71-5300, Thermo Fisher Scientific; IF 1:50), Rab11-FIP1 (#HPA025960, Atlas Antibodies; WB 1:1,000, IF 1:100), RUFY1 (#PA5-31400, Thermo Fisher Scientific; IF 1:100), PARP12 (#HPA063872, Sigma-Aldrich Prestige Antibodies, WB 1:2000); goat polyclonal antibody to PARP12 (#ab45873, Abcam, IF 1:200); HRP-conjugated secondary antibodies (Calbiochem), HRP-conjugated anti-rabbit secondary antibody, heavy Chain Specific (Sigma-Aldrich); AlexaFluor-488,-568 and -647 conjugated secondary antibodies and AlexaFluor -488, -568, -633 or -647 Conjugate transferrins (Thermo Fisher Scientific, Invitrogen).

Techniques: Translocation Assay, Binding Assay, Clinical Proteomics, Membrane

Journal: bioRxiv

Article Title: The endocytic recycling pathway is controlled by the ADP-ribosylated GTPase Rab14

doi: 10.1101/2022.11.26.517555

Figure Lengend Snippet: (a) In vitro ADP-ribosylation assay using GST-tagged purified PARP12 catalytic fragment and His-tagged purified Rab14 wild-type (Rab14 WT) or its MARylation defective mutant E159Q-E162Q (Rab14 MUT), in presence of 4 μCi of [ P]-NAD + and 30 μM of total NAD + . Reactions were stopped at different times (as indicated); the incorporated [ P]-ADP-ribose was detected by autoradiography (AR [ P]). Lower panels show total levels of Rab14 and PARP12. (b) Quantifications of in vitro MARylated Rab14 are reported in the graph. Data represent the mean ± S.D. (N = 3 independent experiments, two-sided one-sample t-test; *P <0,05). (c) Representative Af1521 macro domain pull-down assay of total lysates from HeLa cells transfected with EGFP-tagged Rab14 WT (WT) or its MARylation defective mutants (E159Q, E162Q, E159Q-E162Q). Af1521 bound proteins (Af1521 macro domain pull-down) and total cell lysates (inputs) were separated by SDS-PAGE and analyzed by western blotting with a Rab14 antibody. (d) Quantifications of MARylated Rab14 relative to (c) are reported in the graph. Data represent the mean ± S.D. (N = 3 independent experiments; one way Analysis of Variance; ***P <0,0001). (e) Representative confocal microscopy images of transferrin internalization (Alexa-568-Tfn) in HeLa cells transfected with EGFP-tagged Rab14 WT or its MARylation defective mutant E159Q-E162Q (Rab14 MUT). (f) Quantification of cells showing internalized transferrin into perinuclear recycling endosomes on the total transferrin fluorescence is reported in the graph. Data represent the mean ± S.D. (N = 3 independent experiments; two-sided one-sample t-test; *P <0,0025). (g) Representative confocal microscopy images of double-transferrin internalization in HeLa cells transfected with EGFP-tagged Rab14 WT or its MARylation defective mutant E159Q-E162Q (Rab14 MUT). (h) Representative confocal microscopy images of Alexa-633-Transferrin (Tfn) uptake in HeLa cells upon overexpression of EGFP-tagged Rab14 WT or its MARylation defective mutant E159Q-E162Q (Rab14 MUT). Cells were processed for immunofluorescence and Rab11 localization analyzed using a Rab11 antibody (red). Merged signals are also reported. Scale bars, 10 μM.

Article Snippet: Commercially available antibodies: mouse monoclonal antibodies to Transferrin Receptor/CD71 (clone H68.4; #13-6890, Thermo Fisher Scientific; western blots (WB) 1:3,000; immunofluorescence (IF) 1:200), EEA1 (#610456, BD Transduction Laboratories; WB 1:1,000; IF 1:100), Golgin-97 (CDF4, #A-21270, Thermo Fisher Scientific; IF 1:100); rabbit polyclonal antibodies to Rab4 (#ab13252, Sigma-Aldrich; IF 1:100), Rab14 (#R0656; Sigma-Aldrich, WB 1:2,000, IF 1:100), Rab11 (#71-5300, Thermo Fisher Scientific; IF 1:50), Rab11-FIP1 (#HPA025960, Atlas Antibodies; WB 1:1,000, IF 1:100), RUFY1 (#PA5-31400, Thermo Fisher Scientific; IF 1:100), PARP12 (#HPA063872, Sigma-Aldrich Prestige Antibodies, WB 1:2000); goat polyclonal antibody to PARP12 (#ab45873, Abcam, IF 1:200); HRP-conjugated secondary antibodies (Calbiochem), HRP-conjugated anti-rabbit secondary antibody, heavy Chain Specific (Sigma-Aldrich); AlexaFluor-488,-568 and -647 conjugated secondary antibodies and AlexaFluor -488, -568, -633 or -647 Conjugate transferrins (Thermo Fisher Scientific, Invitrogen).

Techniques: In Vitro, Purification, Mutagenesis, Autoradiography, Pull Down Assay, Transfection, SDS Page, Western Blot, Confocal Microscopy, Fluorescence, Over Expression, Immunofluorescence