Journal: Nature Communications

Article Title: The missense mutation Y65C in PQBP1 causes microcephaly and cognitive deficits through a combination of partial loss-of-function and gain-of-function effects

doi: 10.1038/s41467-025-68202-5

Figure Lengend Snippet: a Venn diagram delineating the overlapping distribution of differential binding proteins across parallel IP-MS datasets. n = 3 mice in each group in each independent experiment. b Top 10 enriched biological processes ranked by P -value. Analysis of the overlapping proteins from ( a ) using a one-sided hypergeometric test with Benjamini–Hochberg FDR correction. Numbers and the size of circles represent counts. c Heatmap visualization reveals differential expression patterns of enhanced binding proteins from ( a ). IBAQ intensities from duplicate IP-MS experiments were quantile-normalized and averaged. d Scatter plot depicting the genes with APA changes (FDR < 0.05) in E12.5 Pqbp1 Y65C/Y cortex. Blue: Transcripts with proximal PAS preference in Y65C. Red: Transcripts demonstrating distal PAS preference in Y65C. n = 3 mice in each group. e The genes with APA changes (FDR < 0.05) in E12.5 Pqbp1-cKO cortex. Blue: transcripts with proximal PAS preference in cKO compared to the control; Red: Transcripts demonstrating distal PAS preference in cKO. n = 3 mice in each group. f Venn diagram shows the overlapped targets between Pqbp1 Y65C/Y and Pqbp1-cKO . g The genes overlaping with known ESC self-renewal regulators. h Integrative Genomics Viewer tracks show the representative gene reads of Marcksl1 from RNA-seq. i qRT-PCR measures the ratios of transcripts with the extended 3′ UTR of Marcksl1 . The locations of primer sets used to amplify specific regions are marked (top). n = 3 mice in each group, p = 0.0329. j CoIP of PQBP1 and FIP1L1. The cortex of mice at E15.5 was immunoprecipitated with anti-PQBP1 antibodies. n = 3 biologically independent experiments, p = 0.0085. k , l Dual-luciferase reporter assays for Flag, Flag-PQBP1, and Flag-PQBP1 Y65C groups. The ratios of R/F (Renilla luciferase 480 nm/firefly luciferase 560 nm) were normalized to the Flag group. CMV, CMV promoter; R luc, Renilla luciferase gene; IRES, internal ribosomal entry site; F luc, firefly luciferase gene. n = 5 biologically independent experiments, Flag vs Flag PQBP1: p < 0.0001; Flag vs Flag PQBP1 Y65C : p = 0.0002. All quantification data are represented as mean ± SD. j Two-tailed unpaired Student’s t test. l Two-tailed one-way ANOVA with Tukey’s multiple comparisons test to adjust for multiple comparisons. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. Source data are provided as a Source Data file.

Article Snippet: Primary antibodies used were PQBP1 (16264-1-AP, Proteintech, 1:1000 for use); RPS3 (ab128995, abcam, 1:1000 for use); RPL13 (ab134961, abcam, 1:1000 for use); FIP1L1 (83863-2-RR, Proteintech, 1:1000 for use); anti-α-tubulin (T9026, Sigma-Aldrich, 1:5000 for use); anti-actin (T0022, Affinity, 1:5000 for use).

Techniques: Binding Assay, Protein-Protein interactions, Quantitative Proteomics, Control, RNA Sequencing, Quantitative RT-PCR, Immunoprecipitation, Luciferase, Two Tailed Test

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: Molecular Medicine

Article Title: The transcription factor Stat-1 is essential for Schwann cell differentiation, myelination and myelin sheath regeneration

doi: 10.1186/s10020-023-00667-w

Figure Lengend Snippet: Stat1 may recruit Rab11fip1 to regulate SC differentiation. A Stat1-binding regions obtained by ChIP-seq. The colored rectangle annotation represent peaks at the promoter (defined as ≤ 1000 bp), the promoter (defined as 1000 − 3000 bp), downstream of thepromoter (defined as ≤ 300 bp), 5’ UTR, 3’UTR, exon, intron or distal intergenic. B Distribution of enrichment intensity of ChIP-seq reads in the proximity of the transcription start site (TSS) (up), and consensus motifs at Stat1 bound sequences (down). C GO enrichment analysis of peak-related genes (bubble diagram). D Venn diagram shows overlap of genes with top 500 peak in the promoter related genes and 386 differentially expressed genes ( p < 0.05 and Fold change ≥ 2) between Stat1 knockdown groups and the control groups. E Histograms visualizing Stat1 binding around the 4 overlapping genes (Ano1, Nts, C1qb and Rab11fip1) locus. “Peak number” represents the plausibility ranking of the peak. F ChIP-qPCR for Stat1 occupancy on the Ano1, Nts, C1qb and Rab11fip1 promoters in differentiating SCs. T -test, ** p < 0.01, *** p < 0.01, n = 3 per group. G Luciferase activity of Ano1, Nts, C1qb and Rab11fip1 promoter and mutant promoter in HEK293 cells co-transfected with pGV141-Stat1 (Stat1 overexpression plasmid) plus an plasmid containing the Renilla luciferase gene. T -test, *** p < 0.01, n = 3 per group. H Western blots showing the expression level of Rab11fip1 and MAG in differentiated SCs treated with Rab11fip1-siRNA and Scramble. The histograms showing that knockdown Rab11fip1 reduces MAG expression in differentiated SCs. T -test, *** p < 0.001 vs Scramble, n = 3 per group

Article Snippet: After blotting onto PVDF membranes (Millipore, Bedford, MA, USA), the membranes were blocked with 5% nonfat milk for 1 h at room temperature and then incubated overnight at 4 °C with the primary antibodies as follow: Stat1 (14994, Cell Signaling, 1:1000), MAG (34-6200, Invitrogen, 1:100), Egr2 (NB110-59723, Novus, 1:500), Nab2 (PA5-75321, ThermoFisher, 1:500), P0 (NB100-1607, Novus, CO, USA; 1:500), Rab11fip1 (sc-517228, Santa Cruz, CA, USA; 1:500) and GAPDH (60004-1-Ig, Proteintech, Chicago, USA; 1:20,000).

Techniques: Binding Assay, ChIP-sequencing, Knockdown, Control, ChIP-qPCR, Luciferase, Activity Assay, Mutagenesis, Transfection, Over Expression, Plasmid Preparation, Western Blot, Expressing

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