Journal: Cell reports

Article Title: p120 RasGAP and ZO-2 are essential for Hippo signaling and tumor-suppressor function mediated by p190A RhoGAP

doi: 10.1016/j.celrep.2023.113486

Figure Lengend Snippet:

Article Snippet: ZO-2 , Boster , Cat# PA1957.

Techniques:

Journal: Cell reports

Article Title: p120 RasGAP and ZO-2 are essential for Hippo signaling and tumor-suppressor function mediated by p190A RhoGAP

doi: 10.1016/j.celrep.2023.113486

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: ZO-2 , Boster , Cat# PA1957.

Techniques: Virus, Recombinant, cDNA Synthesis, RNA Sequencing, Knockdown, Plasmid Preparation, shRNA, Software

Journal: Molecular Biology of the Cell

Article Title: JAM-A associates with ZO-2, afadin, and PDZ-GEF1 to activate Rap2c and regulate epithelial barrier function

doi: 10.1091/mbc.E13-06-0298

Figure Lengend Snippet: JAM-A associates with ZO-2, an important component of barrier function. (A) JAM-A interacts with ZO-2 in vitro. A proteomic array containing 96 PDZ domains from 48 different proteins was used to screen for proteins interacting with the cytoplasmic PDZ-binding motif of JAM-A. The recombinant full-length cytoplasmic tail of JA (JA.CT) directly interacted with the second PDZ domain of ZO-2 on the array but did not detectably interact with any PDZ domains of ZO-1. In contrast, a mutant lacking the PDZ-binding motif on the cytoplasmic tail of JAM-A (JA.CTΔFLV) failed to interact with ZO-2. (B) JAM-A (JA) coimmunoprecipitates with ZO-2 but not ZO-1 in intestinal epithelial cells. JAM-A immunoprecipitates from cell lysates prepared with an NP40-based buffer revealed a 160-kDa ZO-2 immunoreactive band. siRNA down-regulation of ZO-2 was used to confirm specificity of the detected band. (C) Down-regulation of ZO-1, ZO-2, or ZO-1 and -2 led to decreased TER in SK-CO15 cells at similar levels to down-regulation of JAM-A relative to control cells (Scr; n > 5; mean relative resistance with 95% confidence interval). (D) siRNA-mediated down-regulation of ZO-1 and/or ZO-2 was confirmed by immunoblotting.

Article Snippet: Other antibodies were commercially available: polyclonal affinity-purified rabbit anti–JAM-A (Invitrogen); monoclonal mouse Rap2, monoclonal mouse anti–PDZ-GEF1, monoclonal mouse anti-afadin, monoclonal mouse ZO-1, and polyclonal affinity-purified rabbit ZO-2 (BD Transduction Laboratories, Lexington, KY); polyclonal affinity-purified rabbit anti–afadin 02246, monoclonal mouse anti-tubulin, and polyclonal affinity-purified rabbit anti-actin (Sigma-Aldrich, St. Louis, MO); polyclonal affinity-purified rabbit anti-Rap2c (Cell Signaling Technology, Beverly, MA); and polyclonal affinity-purified rabbit anti-RhoA (Santa Cruz Biotechnology).

Techniques: In Vitro, Binding Assay, Recombinant, Mutagenesis, Western Blot

Journal: Molecular Biology of the Cell

Article Title: JAM-A associates with ZO-2, afadin, and PDZ-GEF1 to activate Rap2c and regulate epithelial barrier function

doi: 10.1091/mbc.E13-06-0298

Figure Lengend Snippet: Down-regulation of afadin and PDZ-GEF1, but not PDZ-GEF2 or Rap1a/b, leads to decreased resistance across IECs. (A) Transient down-regulation of afadin (AF) and PDZ-GEF1 (PG1) reduced intestinal epithelial TER to levels comparable to those observed after transient JAM-A down-regulation (JA). Down-regulation of PDZ-GEF2 (PG2), Rap1a (R1a), and Rap1b (R1b) did not affect intestinal epithelial TER ( n > 4; mean relative resistance with 95% confidence interval; data for JAM-A from ). (B) JAM-A (JA) and ZO-2 coimmunoprecipitate with PDZ-GEF1 (PG1) and afadin (AF) in IECs. Cell lysates were preextracted with an NP40-based buffer, and pellets were resuspended in RIPA buffer before coimmunoprecipitation with PDZ-GEF1 or afadin. (C) PDZ-GEF1 localizes to the perijunctional region of IECs. (D) JAM-A (JA) coimmunoprecipitation with afadin (AF) is disrupted after transient down-regulation of ZO-2. Cell lysates were prepared with a Brij97-based buffer before coimmunoprecipitation with afadin.

Article Snippet: Other antibodies were commercially available: polyclonal affinity-purified rabbit anti–JAM-A (Invitrogen); monoclonal mouse Rap2, monoclonal mouse anti–PDZ-GEF1, monoclonal mouse anti-afadin, monoclonal mouse ZO-1, and polyclonal affinity-purified rabbit ZO-2 (BD Transduction Laboratories, Lexington, KY); polyclonal affinity-purified rabbit anti–afadin 02246, monoclonal mouse anti-tubulin, and polyclonal affinity-purified rabbit anti-actin (Sigma-Aldrich, St. Louis, MO); polyclonal affinity-purified rabbit anti-Rap2c (Cell Signaling Technology, Beverly, MA); and polyclonal affinity-purified rabbit anti-RhoA (Santa Cruz Biotechnology).

Techniques:

Journal: Molecular Biology of the Cell

Article Title: JAM-A associates with ZO-2, afadin, and PDZ-GEF1 to activate Rap2c and regulate epithelial barrier function

doi: 10.1091/mbc.E13-06-0298

Figure Lengend Snippet: Rap2 is expressed in the apical spaces between IECs, and Rap2c is involved in the regulation of intestinal epithelial barrier function. (A) mRNAs for Rap2 subtypes Rap2b and Rap2c but not Rap2a are present in SK-CO15 cells, as observed by PCR. RNA was extracted from confluent SK-CO15 cells and subjected to RT-PCR. A common cDNA template and PCR master mix was prepared and then subdivided before addition of Rap2a, Rap2b or Rap2c primers. (B) Rap2 protein is present in apical intercellular junctions of SK-CO15 cells. (C) Rap2 colocalizes with the tight junction marker ZO-2 in colonic mucosa from human pathology specimens. Tissue sections were pretreated with 0.1% Triton X-100 before fixation. (D) qRT-PCR–verified down-regulation of Rap2c (R2c) but not Rap2b (R2b) led to significant reductions in colonic epithelial TER at levels comparable to those observed after transient JAM-A down-regulation (JA; n > 5; relative mean resistance with 95% confidence interval; data for JAM-A from ).

Article Snippet: Other antibodies were commercially available: polyclonal affinity-purified rabbit anti–JAM-A (Invitrogen); monoclonal mouse Rap2, monoclonal mouse anti–PDZ-GEF1, monoclonal mouse anti-afadin, monoclonal mouse ZO-1, and polyclonal affinity-purified rabbit ZO-2 (BD Transduction Laboratories, Lexington, KY); polyclonal affinity-purified rabbit anti–afadin 02246, monoclonal mouse anti-tubulin, and polyclonal affinity-purified rabbit anti-actin (Sigma-Aldrich, St. Louis, MO); polyclonal affinity-purified rabbit anti-Rap2c (Cell Signaling Technology, Beverly, MA); and polyclonal affinity-purified rabbit anti-RhoA (Santa Cruz Biotechnology).

Techniques: Reverse Transcription Polymerase Chain Reaction, Marker, Quantitative RT-PCR

Journal: Molecular Biology of the Cell

Article Title: JAM-A associates with ZO-2, afadin, and PDZ-GEF1 to activate Rap2c and regulate epithelial barrier function

doi: 10.1091/mbc.E13-06-0298

Figure Lengend Snippet: JAM-A down-regulation enhances cytoskeletal contraction. (A) JAM-A − / − mice exhibit increased intestinal permeability to 3-, 10-, and 40 kDa dextran compared to WT mice ( n = 3; bars, mean ± SEM). (B) JAM-A–deficient SK-CO15 cells (shJA) demonstrate enhanced flux to 40-kDa dextran compared with control cells (NS). Dextran flux to the bottom chamber was assessed after 2 h (representative experiment with three independent sample; mean ± SD). (C) Transient down-regulation of ZO-2 in IECs results in enhanced permeability of 40-kDa dextran compared to control (Scr; representative experiment with three independent samples; mean ± SD). (D) Actin turnover rates in control (NS) or JAM-A deficient (shJA) cells are not statistically different, as assessed by FRAP. Stable control (NS) or JAM-A deficient (shJA) cells expressing actin-GFP and grown in chambered wells before assessment of FRAP for actin-GFP at junctions (n = 8; mean ± SEM). (E) Stable down-regulation of JAM-A (shJA) leads to enhanced levels of total and active RhoA as determined by Rhotekin pull-down assay (densitometry calculated as RhoA pull-down signal over total RhoA signal, relative to NS control). (F, G) Stable down-regulation of JAM-A (shJA) leads to enhanced levels of pMLC (S19) as determined by Western blot (F; densitometry calculated as pMLC signal over tubulin signal, relative to NS control) and confocal immunofluorescence staining (G).

Article Snippet: Other antibodies were commercially available: polyclonal affinity-purified rabbit anti–JAM-A (Invitrogen); monoclonal mouse Rap2, monoclonal mouse anti–PDZ-GEF1, monoclonal mouse anti-afadin, monoclonal mouse ZO-1, and polyclonal affinity-purified rabbit ZO-2 (BD Transduction Laboratories, Lexington, KY); polyclonal affinity-purified rabbit anti–afadin 02246, monoclonal mouse anti-tubulin, and polyclonal affinity-purified rabbit anti-actin (Sigma-Aldrich, St. Louis, MO); polyclonal affinity-purified rabbit anti-Rap2c (Cell Signaling Technology, Beverly, MA); and polyclonal affinity-purified rabbit anti-RhoA (Santa Cruz Biotechnology).

Techniques: Permeability, Expressing, Pull Down Assay, Western Blot, Immunofluorescence, Staining

Journal: Molecular Biology of the Cell

Article Title: JAM-A associates with ZO-2, afadin, and PDZ-GEF1 to activate Rap2c and regulate epithelial barrier function

doi: 10.1091/mbc.E13-06-0298

Figure Lengend Snippet: Model of JAM-A–mediated barrier function. We propose that JAM-A is part of a complex composed of ZO-2, afadin, and PDZ-GEF1 (PG1) that recruits and activates Rap2c (R2c) and controls actomyosin contraction via RhoA activation to regulate epithelial barrier function.

Article Snippet: Other antibodies were commercially available: polyclonal affinity-purified rabbit anti–JAM-A (Invitrogen); monoclonal mouse Rap2, monoclonal mouse anti–PDZ-GEF1, monoclonal mouse anti-afadin, monoclonal mouse ZO-1, and polyclonal affinity-purified rabbit ZO-2 (BD Transduction Laboratories, Lexington, KY); polyclonal affinity-purified rabbit anti–afadin 02246, monoclonal mouse anti-tubulin, and polyclonal affinity-purified rabbit anti-actin (Sigma-Aldrich, St. Louis, MO); polyclonal affinity-purified rabbit anti-Rap2c (Cell Signaling Technology, Beverly, MA); and polyclonal affinity-purified rabbit anti-RhoA (Santa Cruz Biotechnology).

Techniques: Activation Assay