rabbit anti human zo2 polyclonal antibody  (Cell Signaling Technology Inc)

Journal: Cell

Article Title: Phase Separation of Zonula Occludens Proteins Drives Formation of Tight Junctions.

doi: 10.1016/j.cell.2019.10.011

Figure Lengend Snippet: Figure 1. Quantification of ZO1 and ZO2 Concentrations and Dynamics in MDCK-II Cells (A) Scheme of the tight-junction (TJ) complex in mammalian epithelial cells. (B) The domain structure of the main TJ scaffolding proteins (ZOs). (C) Quantification of endogenous concentration of ZO1 and ZO2 in the cytoplasm and the TJ of MDCK-II cells. Calibration of NG fluorescence intensity is shown in Figure S1C. The results are summarized in (F) (n = 30 cells, mean ± SD). (D) Quantification of dynamics of NG-ZO1 and NG-ZO2 in the cytoplasm via FCS. Shown are FCS fits from 10 different cells for NG-ZO1 and NG-ZO2. Com- parison of single-particle brightness (cpp) of free NG, NG-ZO1, and NG-ZO2 shows the oligomeric state of NG-ZO1 and NG-ZO2 (5 and 1.5, respectively). The results are summarized in (F). (E) Quantification of ZO1 and ZO2 dynamics at the TJ via FRAP. NG-ZO1 and NG-ZO2 were bleached selectively at the TJ, and recovery was measured at room temperature over time. Kymographs show that ZO1 and ZO2 recovered rapidly from the cytoplasm but not from the adjacent junctional regions. Double exponential fit of normalized and averaged recovery curves of 5 independent measurements (mean ± SD) is shown. (F) Summary of quantitative imaging, FCS, and FRAP. Note that concentration determined by imaging (first row) refers to monomers, whereas concentration determined by FCS (third row) refers to oligomers.

Article Snippet: To detect protein levels by inmmunoblotting, an iBind systemwas used and the following antibodies were used: ZO1 mouse monoclonal (1:750, Invitrogen, 33-9100), ZO2 polyclonal rabbit (1:750, Cell signaling, 2847S).

Techniques: Scaffolding, Concentration Assay, Single Particle, Imaging

Journal: Cell

Article Title: Phase Separation of Zonula Occludens Proteins Drives Formation of Tight Junctions.

doi: 10.1016/j.cell.2019.10.011

Figure Lengend Snippet: Figure 3. Purified ZO Proteins Form Phosphorylation-Sensitive Liquid Condensates In Vitro (A) SDS-PAGE showing purified ZO1-, ZO2-, and ZO3-GFP before cleavage of the MBP tags. (B) Concentration-dependent phase separation into liquid droplets of ZO1, ZO2, and ZO3 in vitro (buffer: 150 mM KCl, 20 mM HEPES [pH 7.4], and 3% PEG-8k). (C) Fusion events of droplets over time indicate liquid-like material properties of ZO condensates (Video S8). (D) Saturation concentrations, i.e., concentration outside of the condensed phase, in vitro (n = 3 experiments, mean ± SD). (E) De-phosphorylation of ZO1, ZO2, and ZO3 by lambda phosphatase promoted phase separation. Phosphorylation of ZO1, ZO2, and ZO3 by CK2 inhibited phase separation. We detected 47 phosphosites (*) after phosphorylation of ZO1 by casein kinase II by using mass spectrometry.

Article Snippet: To detect protein levels by inmmunoblotting, an iBind systemwas used and the following antibodies were used: ZO1 mouse monoclonal (1:750, Invitrogen, 33-9100), ZO2 polyclonal rabbit (1:750, Cell signaling, 2847S).

Techniques: Phospho-proteomics, In Vitro, SDS Page, Concentration Assay, De-Phosphorylation Assay, Mass Spectrometry

Journal: Cell

Article Title: Phase Separation of Zonula Occludens Proteins Drives Formation of Tight Junctions.

doi: 10.1016/j.cell.2019.10.011

Figure Lengend Snippet: Figure 4. Multivalent Protein-Protein Inter- actions Drive Phase Separation of ZO Proteins (A) Scheme of known self-interaction sites of ZO1 and truncations tested. (B) Purified protein truncations of ZO1 (N-terminal, PSG, and C-terminal). (C) Phase-separation assay of truncation mutants of ZO1, ZO2, and ZO3 at 5 mM in vitro (150 mM KCl, 20 mM HEPES [pH 7.4], and 3% PEG-8k). For all ZO homologs, the full-length (FL) protein and the PSG fragment consistently phase separated into liquid-like droplets. The N-terminal and C-terminal fragments did not phase separate. (D) Partitioning assay to determine protein-protein interactions of phase-separated ZO1-FL with fragments of ZO1, ZO2, and ZO3 (n > 10 droplets, mean ± SD). (E) ZO interaction scheme based on the partition- ing assay. Linear sequence of ZO1, ZO2, and ZO3 and their N-terminal, PSG, and C-terminal regions are depicted as two circles to indicate protein- protein interactions between the same homolog (red), between different homologs (green), and for intra-molecular interactions (blue). References to previous interaction studies are indicated by numbers: *1 (Utepbergenov et al., 2006), *2 (Fan- ning et al., 1998), *3 (Wu et al., 2007), and *4,5 (Lye et al., 2010; Spadaro et al., 2017).

Article Snippet: To detect protein levels by inmmunoblotting, an iBind systemwas used and the following antibodies were used: ZO1 mouse monoclonal (1:750, Invitrogen, 33-9100), ZO2 polyclonal rabbit (1:750, Cell signaling, 2847S).

Techniques: In Vitro, Protein-Protein interactions, Sequencing

Journal: Cell

Article Title: Phase Separation of Zonula Occludens Proteins Drives Formation of Tight Junctions.

doi: 10.1016/j.cell.2019.10.011

Figure Lengend Snippet: Figure 5. The Condensed Phase of ZO1 Selectively Sequesters Tight-Junction Proteins (A) Scheme of known interaction sites of ZO1 with tight-junction proteins. (B) In vitro partition assay of tight-junction proteins (clients) into phase-separated ZO1 compartments. The majority of clients partitioned strongly into ZO1 compartments, whereas the control protein (mCherry) did not (n > 10 droplets, mean ± SD). (C) Partitioning of soluble tight-junction proteins labeled with Dendra2 into condensed ZO1-CLIP- TMR droplets in HEK293 cells. Overall, we observed a comparable partitioning of the client proteins as seen in vitro (n > 10 droplets, mean ± SD) See Figure S4B for additional control mea- surements. (D) FRAP measurements of client proteins in vitro showed that interactions between ZO1 and client proteins are transient, as indicated by the fast re- covery of the protein within the ZO1 compartment. (E) Scheme of local enrichment of tight-junction proteins by partitioning into condensed ZO1 compartments. In the condensed state, low-affin- ity binding of TJ proteins to ZO1 and ZO2 is suffi- cient for strong partitioning as a result of the very high local concentration of binding sites.

Article Snippet: To detect protein levels by inmmunoblotting, an iBind systemwas used and the following antibodies were used: ZO1 mouse monoclonal (1:750, Invitrogen, 33-9100), ZO2 polyclonal rabbit (1:750, Cell signaling, 2847S).

Techniques: In Vitro, Control, Labeling, Binding Assay, Concentration Assay

Journal: Cell

Article Title: Phase Separation of Zonula Occludens Proteins Drives Formation of Tight Junctions.

doi: 10.1016/j.cell.2019.10.011

Figure Lengend Snippet: Figure 7. Model of Tight-Junction Formation by Phase Separation of ZO1 and ZO2 (A) Potential induction of ZO phase separation at nascent cell-cell contact sites. ZO is recruited to early adhesion sites via adherens junction (AJ) receptors and adaptor proteins. Membrane recruitment could be sufficient to cross the concentration threshold for phase separation. However, our experiments suggest that ZO1 is self-inhibited by its U6 domain. We refer to the inhibited state as the closed state because the U6 domain is folded back. In order to promote tight-junction strand assembly, ZO1 might need to be opened. Opening might release the self-inhibition and promote phase separation via multimerization of the PSG domain. (B) Formation of ZO-dense compartments causes partitioning of tight-junction-specific proteins, including claudin receptors. The local accumulation and scaffolding of claudins could be sufficient to trigger polymerization and strand formation. As a result of the fluid-like nature of the ZO1 and ZO2 compartments, the coalescence of multiple growing compartments into a continuous belt is facilitated.

Article Snippet: To detect protein levels by inmmunoblotting, an iBind systemwas used and the following antibodies were used: ZO1 mouse monoclonal (1:750, Invitrogen, 33-9100), ZO2 polyclonal rabbit (1:750, Cell signaling, 2847S).

Techniques: Membrane, Concentration Assay, Inhibition, Scaffolding