Journal: PLoS Pathogens

Article Title: Cellular Mechanisms of Alpha Herpesvirus Egress: Live Cell Fluorescence Microscopy of Pseudorabies Virus Exocytosis

doi: 10.1371/journal.ppat.1004535

Figure Lengend Snippet: Cells were transduced to express mCherry-tagged Rab proteins, infected with PRV 486 expressing gM-pHluorin, and imaged at 4.5–5 hr after PRV infection. (A,D,G) The indicated Rab proteins colocalize with gM-pHluorin particle at the moment of exocytosis (yellow circle). Images correspond to . Scale bars represent 2 µm. (B,E,H) Kymographs of indicated Rab protein (red) and gM-pHluorin (green) fluorescence over time. (C,F,I) Ensemble averages of gM-pHluorin (top, green line) and indicated Rab protein (bottom, red line) relative fluorescence. Shaded area represents standard deviation. (A–C) mCherry-Rab6a. Data represent 37 exocytosis events in 4 independent experiments. (D–F) mCherry-Rab8a. Data represent 41 exocytosis events in 3 independent experiments. (G–I) mCherry-Rab11a. Data represent 34 exocytosis events in 3 independent experiments. (J–K) Rab6a is associated with exocytosis of assembled virions containing capsids. Cells were transduced to express mCherry-Rab6a, and co-infected with PRV 950 and PRV 486. (J) Image is a maximum difference projection corresponding to , depicting virus particle exocytosis events over a 13.7 min time course. Exocytosis events associated with Rab6a (blue) containing gM-pHluorin (green) and capsids (red) are indicated (white circles). Scale bar represents 2 µm. (K) Still images from , depicting a single viral exocytosis event. Images correspond to the boxed area in panel B. Scale bar represents 1 µm.

Article Snippet: Rab8a was obtained from M. Nachury , via Addgene (#24898).

Techniques: Infection, Expressing, Fluorescence, Standard Deviation, Virus

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: MiR-199a-3p-regulated alveolar macrophage-derived secretory autophagosomes exacerbate lipopolysaccharide-induced acute respiratory distress syndrome

doi: 10.3389/fcimb.2022.1061790

Figure Lengend Snippet: MiR-199a-3p mimics augmented LPS-induced SAP secretion via the activation of Rab8a in vitro . (A) Western blot analysis demonstrated the Rab8a levels in RAW264.7 cells with the transfection of MiR-199a-3p mimics or inhibitors. (B) Western blot analysis illustrated the efficient siRNA-mediated knockdown of Rab8a in RAW264.7 cells with the transfection of MiR-199a-3p mimics or inhibitors. (C) NTA revealed significant decreases in vesicle levels from RAW264.7 cells with Rab8a knockdown in the MiR-199a-3p mimic and inhibitor groups compared with the control group. (D) Western blot analysis demonstrated significant decreases in LC3-II levels in vesicles from LPS-stimulated RAW264.7 cells in the Rab8a knockdown groups compared with the control group. The experiments were repeated at least three times (n = 3 per group). Each value represents the mean ± SD of three independent experiments. *p < 0.05 vs . the control; #p < 0.05 vs . the LPS control; **p < 0.05 vs . the mimics group without Rab8a knockdown; ***p < 0.05 vs . the inhibitors group without Rab8a knockdown, one-way ANOVA.

Article Snippet: Then, we blocked the membranes with 5% bovine serum albumin (BSA) for 1 h at room temperature and hatched with primary antibodies overnight at 4°C, which included antibodies against F4/80, LC3B, β-actin, Rab8a (1:1000; #6975, #43566,#4970 and #30325, CST), and PAK4 (1:1000, pS474, Absin).

Techniques: Activation Assay, In Vitro, Western Blot, Transfection, Knockdown, Control

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: MiR-199a-3p-regulated alveolar macrophage-derived secretory autophagosomes exacerbate lipopolysaccharide-induced acute respiratory distress syndrome

doi: 10.3389/fcimb.2022.1061790

Figure Lengend Snippet: MiR-199a-3p mimics activate Rab8a by directly reducing PAK4 expression. (A) The predicted MiR-199a-3p binding site on the PAK4 3′-UTRs was determined using target prediction software. (B) Western blot analysis validated the PAK4 expression when cells were transfected with the MiR-199a-3p mimic/inhibitor. (C) Luciferase reporter plasmid assays with wild-type and mutated PAK4 plasmids co-transfected with MiR-199a-3p- or MiR-NC-packaged plasmids. Dual luciferase control vector plasmids acted as NCs. (D) Nanoparticle tracking analysis revealed significant increases in the level of vesicles from PF-3758309-stimulated RAW264.7 cells in the MiR-199a-3p mimic and inhibitor groups compared with the control group. (E) Western blot analysis demonstrated significant increases in LC3-II levels in vesicles from PF-3758309-stimulated RAW264.7 cells in the MiR-199a-3p mimic and inhibitor groups compared with the control group. (F) Western blot analysis revealed the effect of PF-3758309 on increasing Rab8a levels in RAW264.7 cells transfected with MiR-199a-3p mimics and inhibitors. The experiments were repeated at least three times (n = 3 per group). Each value represents the mean ± SD of three independent experiments. *p < 0.05 vs . the plasmid-control group transfected with MiR-199a-3p plasmids; #p < 0.05 vs . the control group; **p < 0.05 vs . the mimics group without PF-3758309; ***p < 0.05 vs . the inhibitors group without PF-3758309, one-way ANOVA.

Article Snippet: Then, we blocked the membranes with 5% bovine serum albumin (BSA) for 1 h at room temperature and hatched with primary antibodies overnight at 4°C, which included antibodies against F4/80, LC3B, β-actin, Rab8a (1:1000; #6975, #43566,#4970 and #30325, CST), and PAK4 (1:1000, pS474, Absin).

Techniques: Expressing, Binding Assay, Software, Western Blot, Transfection, Luciferase, Plasmid Preparation, Control

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Primary Cilia Formation Mediated by Hsa_Circ_0005185/OTUB1/RAB8A Complex Inhibits Prostate Cancer Progression by Suppressing Hedgehog Signaling Pathway.

doi: 10.1002/advs.202411675

Figure Lengend Snippet: Figure 3. circ_0005185 binds to the 196–247 aa region of OTUB1 and the 32–83 aa region of RAB8A. A) Silver staining image of an SDS-PAGE gel displaying the isolation of circ_0005185/protein complexes from the RNA pull-down experiment in DU145 cells; the red box highlights the specific protein bands enriched in the pull-down complex by the circ_0005185 probe compared to the NC probe. B) RNA pull-down assays coupled with western blot analysis confirmed the binding of circ_0005185 to OTUB1 and RAB8A proteins. C,D) RIP experiments utilizing OTUB1 and RAB8A primary antibodies or IgG were performed to assess the enrichment of circ_0005185 with proteins in DU145 and 22RV1 cells. Western blot was used to detect OTUB1 and RAB8A proteins immunoprecipitated by their respective antibodies or IgG. E–F) Interaction profiles of various regions of OTUB1 and RAB8A proteins with circ_0005185 were analyzed using the catRAPID database (http://www.tartaglialab.com/) to gain insights into their binding specificity. G) RIP assays were employed to quantitatively determine the enrichment of circ_0005185 within the 51–102, 101–152, 146–197, and 196–247 amino acid (aa) regions of the OTUB1 protein. H) Similarly, RIP assays were employed to quantitatively assess the enrichment of circ_0005185 within the 32–83, 51–102, 101–152, and 126–177 aa regions of the RAB8A protein. Data are presented as the mean ± SD (*p < 0.05; **p < 0.01; ***p < 0.001, ns, not significant).

Article Snippet: Magna RIPTM RNA Binding Protein Immunoprecipitation Kit (17-701, Merck KGaA, Darmstadt, Germany), OTUB1 primary antibody (Proteintech, Chicago, USA), and RAB8A primary antibody (Proteintech, Chicago, USA) were used to extract protein-bound RNA, and IgG primary antibody was used as a control.

Techniques: Silver Staining, SDS Page, Isolation, Western Blot, Binding Assay, Immunoprecipitation

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Primary Cilia Formation Mediated by Hsa_Circ_0005185/OTUB1/RAB8A Complex Inhibits Prostate Cancer Progression by Suppressing Hedgehog Signaling Pathway.

doi: 10.1002/advs.202411675

Figure Lengend Snippet: Figure 4. circ_0005185 facilitates the deubiquitination of RAB8A by mediating the interaction between OTUB1 and RAB8A. A) Western blot showed that the protein level of RAB8A increased after overexpression of circ_0005185. B) The Co-IP experiment used OTUB1 antibody to verify the binding between RAB8A and OTUB1, which increased after overexpression of circ_0005185. C) Western blot showed that the protein level of RAB8A increased after overexpression of circ_0005185, while knockdown of OTUB1 rescued the level of RAB8A. D) The results of immunofluorescence showed the localization and expression of RAB8A in the control group and circ_0005185 overexpression group. E,F) The ubiquitination level of RAB8A was detected in DU145 and 22RV1 cells using ubiquitination antibodies. Overexpression of circ_0005185 led to decreased ubiquitination of RAB8A, while knockdown of OTUB1 resulted in increased ubiquitination of RAB8A. G,H) The regulation of ubiquitination at the K48 site of RAB8A by OTUB1 was detected in DU145 and 22RV1 cells using antibodies specific to the K48 ubiquitination site. I) Ubiquitination at the K63 site of RAB8A was detected in DU145 cells using antibodies specific to the K63 ubiquitination site.

Article Snippet: Magna RIPTM RNA Binding Protein Immunoprecipitation Kit (17-701, Merck KGaA, Darmstadt, Germany), OTUB1 primary antibody (Proteintech, Chicago, USA), and RAB8A primary antibody (Proteintech, Chicago, USA) were used to extract protein-bound RNA, and IgG primary antibody was used as a control.

Techniques: Western Blot, Over Expression, Co-Immunoprecipitation Assay, Binding Assay, Knockdown, Expressing, Control, Ubiquitin Proteomics

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Primary Cilia Formation Mediated by Hsa_Circ_0005185/OTUB1/RAB8A Complex Inhibits Prostate Cancer Progression by Suppressing Hedgehog Signaling Pathway.

doi: 10.1002/advs.202411675

Figure Lengend Snippet: Figure 6. circ_0005185 inhibits the progression of prostate cancer in vivo. A) Images of subcutaneous xenografted tumor in the nude mice vector group and circ_0005185-OE group. B) Tumor volume measured every 5 days. C) Weight of xenograft. D) qRT-PCR showed that the expression of circ_0005185 in the tumors from the mice of the overexpression group was significantly higher than that in the control group. E–G) IHC results of RAB8A, CCND1, and c-MYC in xenografts. Data are presented as the mean ± SD (*p < 0.05; ***p < 0.001; scale bar = 50 and 25 μm).

Article Snippet: Magna RIPTM RNA Binding Protein Immunoprecipitation Kit (17-701, Merck KGaA, Darmstadt, Germany), OTUB1 primary antibody (Proteintech, Chicago, USA), and RAB8A primary antibody (Proteintech, Chicago, USA) were used to extract protein-bound RNA, and IgG primary antibody was used as a control.

Techniques: In Vivo, Plasmid Preparation, Quantitative RT-PCR, Expressing, Over Expression, Control

Journal: Journal of Neuroscience

Article Title: Retinal Degeneration and Failure of Photoreceptor Outer Segment Formation in Mice with Targeted Deletion of the Joubert Syndrome Gene, Ahi1

doi: 10.1523/jneurosci.5229-09.2010

Figure Lengend Snippet: Figure 8. Reduced Rab8a expression in retinal photoreceptors from day E18.5 Ahi1/ mice. A, Retinas from Ahi1/ and Ahi1/ mice were immunostained for the membrane trafficking protein,Rab8a(green),andwerecolabeledwithrhodopsin(red).Rab8aandrhodopsincolocalizedin immature photoreceptor cells, in both Ahi1/ and Ahi1/ mice. The same exposure settings wereusedfortheAhi1/andAhi1 /retinas.DNAisvisualizedwithDAPI(blue).Scale bar, 20 m. B, Lower-magnification images of Rab8a (red) immunostaining in retinal photoreceptors from Ahi1/ and Ahi1 / mice. Much weaker Rab8a immunostaining was observed in Ahi1 / photoreceptors (right) than in Ahi1/ photoreceptors (left). The same exposure settings were used for the Ahi1/ and Ahi1 / retinas. DNA is visualized with DAPI (blue). Scale bar, 50 m.

Article Snippet: The following primary antibodies were used for immunostaining at the indicated final dilutions: Ahi1 (rabbit IgG; 1:1000) (Doering et al., 2008); rhodopsin (mouse IgG, clone 4D2, 1:1000, kindly provided by Dr. R. S. Molday, University of British Columbia, Vancouver, Canada; and mouse IgG, clone 1D4, 1:1000, Sigma) (Molday and MacKenzie, 1983); synaptotagmin (mouse IgG2a, 1:100; Calbiochem) (Matthew et al., 1981); anti- - tubulin (mouse IgG1, 1:300; Sigma) (Hsiao et al., 2009); Rab8a (mouse IgG2b, 1:50; BD Biosciences) (Hsiao et al., 2009); -transducin (rabbit IgG, 1:1000; Santa Cruz Biotechnology) (Chen et al., 2007); Rom1 (mouse IgG, 1D5, 1:40; kindly provided by Dr. R. S. Molday) (Bascom et al., 1992); Rp1 (chicken IgY, 1:2000) (Liu et al., 2002); Rpgrip1 (rabbit IgG, 1:5000; kindly provided by Dr. Tiansen Li, Massachussetts Eye and Ear Infirmary, Boston, MA) (Hong et al., 2001); and anti-cyclic nucleotide gated channel 1 (mouse IgG, 1D1, 1:10; kindly provided by Dr. R. S. Molday) (Cook et al., 1989).

Techniques: Expressing, Membrane, Immunostaining

Journal: EMBO Reports

Article Title: TMEM135 regulates primary ciliogenesis through modulation of intracellular cholesterol distribution

doi: 10.15252/embr.201948901

Figure Lengend Snippet: RPE1 cells were transfected with siRNAs as indicated, followed by transfection with wild‐type pGFP‐Rab8a (WT‐Rab8), constitutively active pGFP‐Rab8a (Q67L) (CA‐Rab8), or DN Rab8 dominant‐negative pGFP Rab8a (T22N), incubated in serum‐starved media for 12 h, and immunostained for ARL13B (red), GFP‐Rab8 (green), and DAPI (blue). Scale bar, 10 μm. Quantification of the percentage of ciliated cells shown in (A). Data represent mean ± SD ( n = 3 experiments), and 200 GFP‐positive cells were scored per condition per experiment; * P < 0.05, Student's t ‐test. (Upper panel) Cells were transfected as indicated and the cell lysates were incubated with purified proteins, including GST or GST‐JCF1 (RBD). The amount of GTP‐Rab8 bound to GFT‐JCF1(RBD) was analyzed by Western blot with Rab8 antibody. (Lower panel) Intensity of the bands was quantified by ImageJ software. The amount of GTP‐Rab8 was normalized to the control level. Bar graph represents mean ± SD ( n = 3 experiments). * P < 0.05, Student's t ‐test. Cells were transfected as shown in (A), and cell lysates were incubated with purified GST‐JCF1 (RBD) fusion protein. The amount of GTP‐Rab8 bound to GFT‐JCF1(RBD) was analyzed by Western blot with Rab8 antibody. (Lower panel) Cells were transfected as shown in (A), and cell lysates were incubated with purified GST protein. The amount of GTP‐Rab8 bound to GFT‐JCF1(RBD) was analyzed by Western blot with Rab8 antibody. Source data are available online for this figure.

Article Snippet: Human wild‐type pGFP‐Rab8A (Plasmid #24898), human constitutively active (Q67L) pGFP‐Rab8A (Plasmid #24900), and human dominant‐negative (T22N) pGFP‐Rab8A (Plasmid #24899) were obtained from Addgene.

Techniques: Transfection, Dominant Negative Mutation, Incubation, Purification, Western Blot, Software, Control

Journal: EMBO Reports

Article Title: TMEM135 regulates primary ciliogenesis through modulation of intracellular cholesterol distribution

doi: 10.15252/embr.201948901

Figure Lengend Snippet: RPE1 cells were transfected with siRNAs as indicated, followed by 24‐h incubation in serum‐starved media. Cells were then and subjected to fractionation, and Western blot for IFT20, the Golgi marker GM130, membrane marker UBXD8, and nuclear marker CREB. Efficiency of IFT20 knockdown by Western blot. Cells were transfected with siRNAs as indicated, followed by incubation in serum‐starved media for 24 h, and immunostained for ARL13B (red). Scale bar, 10 μm. The bar graph represents the quantification of the percentage of ciliated cells. Data represent mean ± SD ( n = 3 experiments), and 250 cells were scored per condition per experiment; * P < 0.05, Student's t ‐test. Cells were transfected as shown in (C), and immunostained for Rab8 and γ‐tubulin, followed by quantification of the percentage of cells with Rab8 localized to the centriole. Data represent average ( n = 2 experiments). Cells were transfected with siRNAs as indicated, followed by transfection with Flag‐IFT20, incubated in serum‐starvation media for 12 h, and immunostained for ARL13B. Representative fluorescent images of Flag‐IFT20 (green), ARL13B (red), and DAPI (blue) are shown. Scale bar, 10 μm. Quantification of the percentage of ciliated cells with both the Flag‐IFT20 and ARL13B localized in the cilium. Data represent mean ± SD ( n = 3 experiments), and 150 Flag‐positive cells were scored per condition per experiment; * P < 0.05, Student's t ‐test. Cells were transfected with siRNAs as indicated, followed by further transfection with CA‐Rab8, incubated in serum‐starvation media for 12 h, and immunostained for acetylated tubulin. Representative fluorescent images of GFP‐Rab8 Q67L (green), acetylated tubulin (red), and DNA (blue) are shown. Scale bar, 10 μm. Quantification of the percentage of GFP‐positive ciliated cells (only those cilia having both GFP‐Rab8 and acetylated tubulin on cilium were considered for quantification). Data represent mean ± SD ( n = 3 experiments), and 200 GFP‐positive cells were scored per condition per experiment, * P < 0.05, Student's t ‐test. Source data are available online for this figure.

Article Snippet: Human wild‐type pGFP‐Rab8A (Plasmid #24898), human constitutively active (Q67L) pGFP‐Rab8A (Plasmid #24900), and human dominant‐negative (T22N) pGFP‐Rab8A (Plasmid #24899) were obtained from Addgene.

Techniques: Transfection, Incubation, Fractionation, Western Blot, Marker, Membrane, Knockdown

Journal: EMBO Reports

Article Title: TMEM135 regulates primary ciliogenesis through modulation of intracellular cholesterol distribution

doi: 10.15252/embr.201948901

Figure Lengend Snippet: A Efficiency of Rab8a depletion confirmed by Western blot in RPE1 cells. B RPE1 cells were transfected by siRNAs as indicated, followed by incubation in serum‐starvation media for 24 h, and immunostained for ARL13B (red) and γ‐tubulin (green). Scale bar, 10 μm. C Quantification of the percentage of ciliated cells shown in (B). Data represent mean ± SD ( n = 3 experiments), and 250 GFP‐positive cells were scored per condition per experiment; * P < 0.05, Student's t ‐test. D Cells were transfected by siRNA as indicated followed by incubation in serum‐starvation media for 24 h, and immunostained for EHD1 (red) and γ‐tubulin (green). Scale bar, 10 μm. E Quantification of the percentage of cells with EHD1 in cilium or in the distal end of basal body as shown in (D). Data represent mean ± SD ( n = 3 experiments), and 150 cells were scored per condition per experiment; * P < 0.05, Student's t ‐test. F Cells were transfected by siRNAs as indicated, followed by incubation in serum‐starvation media for 24 h, and immunostained for IFT20 (red) and γ‐tubulin (green). Scale bar, 10 μm. G Quantification of the percentage of IFT20 fluorescent intensity at the centriole shown in (F). Data represent mean ± SD ( n = 3 experiments), and 150 were scored per condition per experiment; * P < 0.05, Student's t ‐test. H–J Cells were transfected by siRNAs as indicated, followed by transfection with GFP‐Rab8 WT, GFP‐Rab8 Q67L, or GFP‐Rab8 T22N, and further incubated in serum‐starvation media for 12 h. Cell lysate was subjected to immunoprecipitation with anti‐GFP antibody, followed by Western blot with antibody against GFP. Source data are available online for this figure.

Article Snippet: Human wild‐type pGFP‐Rab8A (Plasmid #24898), human constitutively active (Q67L) pGFP‐Rab8A (Plasmid #24900), and human dominant‐negative (T22N) pGFP‐Rab8A (Plasmid #24899) were obtained from Addgene.

Techniques: Western Blot, Transfection, Incubation, Immunoprecipitation

Journal: iScience

Article Title: Pathogenic LRRK2 regulates centrosome cohesion via Rab10/RILPL1-mediated CDK5RAP2 displacement

doi: 10.1016/j.isci.2022.104476

Figure Lengend Snippet: LRRK2-mediated centrosomal cohesion deficits depend on both GTP conformation and phosphorylation status of Rab8a (A) Example of HEK293T cells co-transfected with FLAG-tagged wild-type LRRK2 and GFP-tagged Rab8a-Q67L (GTP-trapped) or GFP-tagged Rab8a-Q67L-T72A (GTP-trapped but non-phosphorylatable), and stained with antibody against FLAG (red) and the centrosomal marker pericentrin (pseudocolored blue) and DAPI. Arrows point to centrosomes in transfected cells. Cell boundaries (yellow) are shown and were determined by FLAG staining because of FLAG-tagged wild-type LRRK2 expression (or GFP because of GFP-tagged Rab8a expression for single transfection experiments). Scale bar, 10 μm. (B) Quantification of the split centrosome phenotype in cells expressing the indicated constructs, in either the absence or presence of LRRK2 kinase inhibitor MLi2 (100 nM, 2 h) as indicated. Around 100–150 cells were quantified per condition and experiment. Bars represent mean ± SEM (n = two to three independent experiments; wt-LRRK2 versus wt-LRRK2 + Rab8a, p = 0.007; wt-LRRK2 + Rab8a versus wt-LRRK2 + Rab8a + MLi2, p = 0.024; wt-LRRK2 versus wt-LRRK2 + Rab8a-Q67L, p = 0.003; wt-LRRK2 + Rab8a-Q67L versus wt-LRRK2 + Rab8a-Q67L + MLi2, p = 0.026); ∗∗∗p < 0.005; ∗∗p < 0.01; ∗p < 0.05. (C) Cells were co-transfected with FLAG-tagged wild-type LRRK2 and GFP-tagged Rab8a constructs as indicated, left untreated or incubated with 100 nM MLi2 for 2 h, and extracts blotted for FLAG-tagged LRRK2 (FLAG-LRRK2), FLAG-tagged S935-phosphorylated LRRK2 (FLAG-pS935-LRRK2) as a readout for on-target effect of MLi2, phosphorylated GFP-tagged Rab8a (GFP-pT72-Rab8a), total GFP-tagged Rab8a (GFP-Rab8a), and tubulin or GAPDH as loading controls. (D) HeLa cells were transfected with the indicated GFP-tagged Rab8a constructs, briefly fixed, washed and stained with DAPI. All GFP-tagged Rab8a constructs except the inactive T22N mutant display a localization consistent with their presence in a tubular early recycling compartment. Scale bar, 10 μm.

Article Snippet: pGFP-hRab8a T22N , , Addgene Cat#24899.

Techniques: Phospho-proteomics, Transfection, Staining, Marker, Expressing, Construct, Incubation, Mutagenesis

Journal: iScience

Article Title: Pathogenic LRRK2 regulates centrosome cohesion via Rab10/RILPL1-mediated CDK5RAP2 displacement

doi: 10.1016/j.isci.2022.104476

Figure Lengend Snippet:

Article Snippet: pGFP-hRab8a T22N , , Addgene Cat#24899.

Techniques: Virus, Recombinant, Plasmid Preparation, Mutagenesis, In Situ, Knock-In, Control, Enzyme-linked Immunosorbent Assay

Journal: Molecular Biology of the Cell

Article Title: The RabGAPs EPI64A and EPI64B regulate the apical structure of epithelial cells ^†

doi: 10.1091/mbc.e21-05-0268

Figure Lengend Snippet: FIGURE 1: EPI64A and EPI64B both localize to apical microvilli. (A) Schematic of EPI64A and EPI64B domains with percentage identities and the TBC/RabGAP arginine residue necessary for the RabGAP activity indicated. (B) Western blots with antibodies to EPI64B and tubulin of Jeg-3 cell lysates after treatment with the indicated siRNAs to EPI64B. (C) Western blot with antibodies to EPI64B on cell lysates from several cultured cell lines. (D) SIM microscopy of Jeg-3 cells transfected with GFP-EPI64A (top block of panels) or GFP-EPI64B (bottom block of panels). Cells were transfected to express GFP-tagged constructs (green) and stained for ezrin (red) and actin (blue). Arrows indicate the localization of GFP-EPI64A to the base of microvilli. Scale bars: top panels, 10 µm; bottom panels, 10 µm.(E) Quantitation of GFP- EPI64A and GFP-EPI64B to localize to microvilli in wild-type cells.

Article Snippet: GFP-Rab8a wild type was cloned in the lab; EGFP-Rab8a-TN (Addgene #86077) and EGFP-Rab8a-QL (Addgene #86076) were gifts from Lei Liu.

Techniques: Residue, Activity Assay, Western Blot, Cell Culture, Microscopy, Transfection, Blocking Assay, Construct, Staining, Quantitation Assay

Journal: Molecular Biology of the Cell

Article Title: The RabGAPs EPI64A and EPI64B regulate the apical structure of epithelial cells ^†

doi: 10.1091/mbc.e21-05-0268

Figure Lengend Snippet: FIGURE 2: EPI64A and EPI64B can localize to microvilli independently of the scaffolding protein EBP50 (A) Jeg-3 cells transfected with 3xFLAG-EBP50 were co-transfected with either GFP-EPI64A or GFP-EPI64B. The GFP-tagged proteins were immunoprecipitated with GFP-Trap beads and the immunoprecipitates blotted for FLAG and GFP. (B) Confocal imaging of microvillar localization of GFP-EPI-64A and GFP-EPI64A-LA, which cannot bind EBP50, in Jeg-3 cells. Scale bar 10 µm. (C) Western blot of cell lysates of Jeg-3 wild type, or CRISPR-modified EBP50 deletion cell line, blotted for ezrin, EBP50, and tubulin. Scale bar: 10 µm (D) Localization of ezrin and actin in Jeg-3 cells lacking endogenous EBP50. Scale bars: 10 µm. (E) Confocal imaging of GFP-EPI64A or GFP-EPI64B in Jeg-3 cells lacking EBP50. Scale bars: 10 µm.

Article Snippet: GFP-Rab8a wild type was cloned in the lab; EGFP-Rab8a-TN (Addgene #86077) and EGFP-Rab8a-QL (Addgene #86076) were gifts from Lei Liu.

Techniques: Scaffolding, Transfection, Immunoprecipitation, Imaging, Western Blot, CRISPR, Modification

Journal: Molecular Biology of the Cell

Article Title: The RabGAPs EPI64A and EPI64B regulate the apical structure of epithelial cells ^†

doi: 10.1091/mbc.e21-05-0268

Figure Lengend Snippet: FIGURE 3: EPI64A contains a localization domain spanning its TBC domain. (A) Schematic of the EPI64 constructs used in B and C and summary of results shown in this figure (B) Confocal imaging of GFP-EPI64A-314-508, which contains the C-terminal -DTYL sequence, in Jeg-3 wild-type cells and Jeg-3 cells lacking EBP50. (C) Confocal images showing the localization of GFP-tagged deletion constructs of GFP-EPI64A. Scale bar 10 µm. (D) Immunolocalization of two HA-tagged constructs, the top one containing the minimal region that localizes to microvilli (HA-EPI64A-61-408) and the bottom one (HA-71-408) that does not localize. Scale bar: 10 µm. (E) GFP-trap pull down: Jeg-3 cells were transfected with either GFP or GFP-Arf6 together with the indicated HA-EPI64A constructs. The GFP or GFP-Arf6 were recovered and analyzed for the presence of the HA-EPI64A constructs by immunoblotting.

Article Snippet: GFP-Rab8a wild type was cloned in the lab; EGFP-Rab8a-TN (Addgene #86077) and EGFP-Rab8a-QL (Addgene #86076) were gifts from Lei Liu.

Techniques: Construct, Imaging, Sequencing, Transfection, Western Blot

Journal: Molecular Biology of the Cell

Article Title: The RabGAPs EPI64A and EPI64B regulate the apical structure of epithelial cells ^†

doi: 10.1091/mbc.e21-05-0268

Figure Lengend Snippet: FIGURE 5: Jeg-3 cells lacking EPI64A and EPI64B lack microvilli (A) Western blot with antibodies to EPI64A, EPI64B, and tubulin on whole cell lysates of Jeg-3 cells genetically modified to lack EPI64A, EPI64B, or both proteins (DKO). (B) Confocal imaging showing localization of ezrin and actin in wild-type Jeg-3 cells and the single (A-KO, B-KO) and double knockout cells. Scale bar 10 µm. (C) Quantitation of the percentage of indicated cells stained for ezrin that express surface microvilli. Normal defined >50% coverage of the apical surface with microvilli. One-way analysis of variance gave the indicated p values. (D) EPI64A/B double knockout cells were transfected to express the indicated constructs and the percentage of ezrin-stained cells (total for either untransfected or GFP-expressing for transfected cells) that express normal apical microvilli. One-way analysis of variance gave the indicated p values. (E) Localization of tight junction ZO-1 in wild-type and knockout Jeg-3 cells. Scale bar: 10 µm.

Article Snippet: GFP-Rab8a wild type was cloned in the lab; EGFP-Rab8a-TN (Addgene #86077) and EGFP-Rab8a-QL (Addgene #86076) were gifts from Lei Liu.

Techniques: Western Blot, Genetically Modified, Imaging, Double Knockout, Quantitation Assay, Staining, Transfection, Construct, Expressing, Knock-Out

Journal: Molecular Biology of the Cell

Article Title: The RabGAPs EPI64A and EPI64B regulate the apical structure of epithelial cells ^†

doi: 10.1091/mbc.e21-05-0268

Figure Lengend Snippet: FIGURE 6: Dominant negative Rab8A and Rab35A can restore microvilli to EPI64A/B double knockout cells. (A) Wild-type Jeg-3 cells were transfected to express GFP or the indicated GFP-Rab proteins and the percentage of cells (total for either untransfected or GFP-expressing for transfected cells) determined that express apical microvilli. (B) Jeg-3 EPI64A/B double knockout cells were transfected with GFP or the indicated GFP-Rab proteins and the percentage of cells (total for either untransfected or GFP-expressing for transfected cells) expressing microvilli scored. One-way analysis of variance gave the indicated p values.

Article Snippet: GFP-Rab8a wild type was cloned in the lab; EGFP-Rab8a-TN (Addgene #86077) and EGFP-Rab8a-QL (Addgene #86076) were gifts from Lei Liu.

Techniques: Dominant Negative Mutation, Double Knockout, Transfection, Expressing

Journal: Molecular Biology of the Cell

Article Title: The RabGAPs EPI64A and EPI64B regulate the apical structure of epithelial cells ^†

doi: 10.1091/mbc.e21-05-0268

Figure Lengend Snippet: FIGURE 7: Caco-2 cells lacking EPI64A and EPI64B have microvilli (A) Western blots of whole cell lysates from Caco-2 BBE1 wild-type, EPI64A, and EPI64B single knockout and double knockout cells blotted for EPI64A, EPI64B, and tubulin. (B) Confocal imaging showing localization of ezrin in the apical region of wild-type and knockout cells. Scale bar: 10 µm. (C) Localization of GFP-EPI64A and GFP-EPI64B expressed in double knockout cells. Scale bar 10 µm.

Article Snippet: GFP-Rab8a wild type was cloned in the lab; EGFP-Rab8a-TN (Addgene #86077) and EGFP-Rab8a-QL (Addgene #86076) were gifts from Lei Liu.

Techniques: Western Blot, Knock-Out, Double Knockout, Imaging

Journal: Molecular Biology of the Cell

Article Title: The RabGAPs EPI64A and EPI64B regulate the apical structure of epithelial cells ^†

doi: 10.1091/mbc.e21-05-0268

Figure Lengend Snippet: FIGURE 8: Caco-2 cells lacking EPI64A and EPI64B have aberrant apical junctions (A) Fields of wild-type, EPI64A and EPI64B single knockout and EPI64A/B double knockout cells stained for actin and the tight junction marker ZO-1. The phenotypes seen were variable, so the most wild–type-looking regions of cells are shown (Normal) and contrasted with regions where the normal polygonal organization is disrupted (Severe). Scale bar: 10 µm. (B) Percentage of wild-type and knockout cells in which one or more of its junctions shows a reflex angle (>180°). One-way analysis of variance gave the indicated p values. (C) Example of stellate knockout cell stained for ezrin, myosin IIA, and actin XY-dimensions (top panels) and YZ-dimensions (bottom panel). (D) Localization of vinculin, actin, and myosin IIA in the apical (top panels) and basal (bottom panels) sections of wild-type and double knockout Caco-2 cells. Scale bars 10 µm.

Article Snippet: GFP-Rab8a wild type was cloned in the lab; EGFP-Rab8a-TN (Addgene #86077) and EGFP-Rab8a-QL (Addgene #86076) were gifts from Lei Liu.

Techniques: Knock-Out, Double Knockout, Staining, Marker