Journal: Cancer Research

Article Title: CircFOXK2 Promotes Growth and Metastasis of Pancreatic Ductal Adenocarcinoma by Complexing with RNA-Binding Proteins and Sponging MiR-942

doi: 10.1158/0008-5472.can-19-3268

Figure Lengend Snippet: Figure 6. Identification of circFOXK2-interacting proteins in PDAC. A, Schematic diagram showing the process of circFOXK2-pulldown. circFOXK2, FOXK2, and circGFP probes were generated by in vitro transcription with biotin-UTP. The biotin-labeled probes were used to pull down interacting proteins in PANC1 cells. The pulldown proteins were identified by mass spectrometry. B, Mass spectrometry analysis of circFOXK2-interacting proteins in PANC1 cells. C, RNA immunoprecipitation for YBX1, hnRNPK, SEPT11, ILF3, RAB11FIP1, and ASF in PDAC cells. The interactions between circFOXK2 and YBX1, hnRNPK, SEPT11, ILF3, RABIIFIP1, and ASF were analyzed by qRT-PCR. D, Expression levels of YBX1 and hnRNPK targets CORO1C, NUF2, PDXK, and PPP2R2C after knockdown of circFOXK2 in PDAC cells. E, circFOXK2- pulldown assay in PANC1 cells using biotin-labeled circFOXK2 probe. The interaction between circFOXK2, NUF2, and PDXK was analyzed by qRT-PCR. F, The expressions of NUF2 and PDXK in circFOXK2-overexpressed HPDE cells after knockdown of YBX1 and hnRNPK. G and H, RNA immunoprecipitation for YBX1 (G) and hnRNPK (H) after knockdown of circFOXK2 in PANC1 cells. The NUF2 and PDXK enrichments were analyzed by qRT-PCR. I, Expression levels of PDXK and NUF2 and the correlations between NUF2 and circFOXK2 level in PDAC primary tumors. Data are from at least three independent experiments. Mean SD. , P < 0.05; , P < 0.01; , P < 0.001.

Article Snippet: Magnetic beads were washed twice with RIP wash buffer, followed by incubation with 2 mg antibody against hnRNPK (rabbit; Proteintech 11426-1-AP), YBX1 (rabbit; Proteintech 20339-1-AP), SEPT11 (rabbit; Proteintech 14672-1-AP), ILF3 (rabbit; Proteintech 19887-1-AP), ASF (rabbit; Proteintech 12929-2-AP), and RAB11FIP1 (rabbit; Proteintech 16778-1-AP) for 30 minutes at room temperature.

Techniques: Generated, In Vitro, Labeling, Mass Spectrometry, RNA Immunoprecipitation, Quantitative RT-PCR, Expressing, Knockdown

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: Construct encoding the human RAB11 family interacting protein 1 (class I), transcript variant 1, (RAB11-FIP1C) (NM_025151) was purchased from OriGene (#RC209640).

Techniques: Translocation Assay, Binding 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: (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: Construct encoding the human RAB11 family interacting protein 1 (class I), transcript variant 1, (RAB11-FIP1C) (NM_025151) was purchased from OriGene (#RC209640).

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

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: Representative confocal microscopy images of transferrin internalization (Alexa-568/633-labeled transferrin, Tnf, as indicated) in HeLa cells transfected with EGFP-tagged Rab14 WT or Rab14 MARylation defective mutant E159Q-E162Q (Rab14 MUT). Cells were processed for immunofluorescence and stained with antibodies against (a) RUFY1 or (b) FIP1c/RCP. Zoom 1 e 2: enlarged view of merged signals. Scale bars 10 μM.

Article Snippet: Construct encoding the human RAB11 family interacting protein 1 (class I), transcript variant 1, (RAB11-FIP1C) (NM_025151) was purchased from OriGene (#RC209640).

Techniques: Confocal Microscopy, Labeling, Transfection, Mutagenesis, Immunofluorescence, Staining

Journal: Molecular cell

Article Title: Molecular mechanisms for CFIm-mediated regulation of mRNA alternative polyadenylation

doi: 10.1016/j.molcel.2017.11.031

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Fip1-RD peptide , GenScript , Custom synthesis: SC1208/U2711BI160_1.

Techniques: Recombinant, Reporter Assay, Transfection, Cloning, In Vitro, Software