rabbit anti p nf2  (Cell Signaling Technology Inc)

Journal: iScience

Article Title: JAM-A signals through the Hippo pathway to regulate intestinal epithelial proliferation

doi: 10.1016/j.isci.2022.104316

Figure Lengend Snippet: JAM-A regulates proliferation and associates with Hippo regulatory molecules LATS1 and NF2 (A) JAM-A-deficient mice exhibit increased colonic IEC proliferation. Representative confocal images of colon tissue from JAM-A ERΔIEC and control ( JAM-A fl/fl ) mice stained for JAM-A (green), Ki67 (red), and DAPI/nuclei (blue). Increased levels of proliferating cells in the JAM-A ERΔIEC mice compared with controls is shown in the graph plotting percentage of Ki67 positive cells/crypt with dots denoting individual crypts. Data are means ± SEM of crypts from JAM-A fl/fl and JAM-A ERΔIEC mice, respectively. ∗∗∗p ≤ 0.001; two-tailed Student’s t test. Scale bar, 50 μm. (B) EdU incorporation in 3D enteroids derived from JAM-A −/− and WT ( JAM-A +/+ ) mice indicate that loss of JAM-A results in increased proliferation. Data are means ± SEM where buds counted from WT and JAM-A −/− enteroids and are denoted as dots on the graph. ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001: two-tailed Student’s t test. Scale bar, 50 μm. (C) Co-immunoprecipitation of ZO-2 and JAM-A with NF2. Whole-cell lysate of SKCO-15 IEC stably expressing a full-length HA-tagged JAM-A (HA-JAM-A) was immunoprecipitated with a ZO-2 (ZO-2 IP) or HA (HA IP) antibody followed by immunoblotting for NF2, ZO-2 and NF2, p-NF2 S518, and HA-JAM-A. p-NF2 S518 did not co-immunoprecipitate with HA-JAM-A. (D) Representative immunostaining of NF2 (green) and JAM-A (red) detected by confocal microscopy in WT murine colon tissue demonstrate co-localization at the TJ. Scale bar, 50 μm. (E) JAM-A co-immunoprecipitates with multiple Hippo pathway molecules. Immunoprecipitation of HA-tagged JAM-A from IEC lysates with an HA-specific antibody (HA IP) followed by western blot LATS1, YAP, p-YAP S127, 14-3-3, and JAM-A. Co-immunoprecipitation of these molecules with JAM-A is demonstrated. (F) Endogenous JAM-A co-immunoprecipitation with LATS1, NF2, and YAP. Immunoprecipitation of LATS1 with LATS1 antibody (LATS1 IP) from IEC cell lysates followed by western blot for NF2, YAP, JAM-A, and LATS1. Co-immunoprecipitation is observed between these molecules. (G) Representative immunofluorescence confocal images of LATS1 (green), JAM-A (red), and DAPI/nuclei (blue) in WT murine colon tissue. Scale bar, 50 μm. Lysate blots serve as input loading controls in (C), (E), and (F). Immunoprecipitation with a nonspecific IgG antibody served as a negative control (IgG IP) for (C), (E), and (F). All data are representative of three independent experiments. See also Figure S1 .

Article Snippet: Rabbit Anti-p-NF2/Merlin S518 , Cell Signaling Technology , 9163; RRID: AB_2149793.

Techniques: Control, Staining, Two Tailed Test, Derivative Assay, Immunoprecipitation, Stable Transfection, Expressing, Western Blot, Immunostaining, Confocal Microscopy, Immunofluorescence, Negative Control

Journal: iScience

Article Title: JAM-A signals through the Hippo pathway to regulate intestinal epithelial proliferation

doi: 10.1016/j.isci.2022.104316

Figure Lengend Snippet: JAM-A is a regulator of Hippo signaling in primary cultured IEC (A) Representative confocal immunostaining of NF2 (green) and JAM-A (red) in WT murine 2D colonoids shows co-localization at the TJ. Scale bar, 20 μm. (B) Representative confocal immunostaining of LATS1 (green) and JAM-A (red) associating at the TJ in WT 2D murine colonoids. Scale bar, 20 μm. (C) JAM-A ERΔIEC primary IEC exhibit prolonged nuclear YAP localization. Representative immunofluorescent images of YAP (green) and ZO-1 (red) in 2D colonoids cultured at low and high cell confluency derived from and JAM-A ERΔIEC and JAM-A fl/fl mice. At low cell confluency YAP staining is visible in the nuclei of JAM-A ERΔIEC and JAM-A fl/fl IEC. At high cell confluency, YAP localizes to the cytoplasm in control IEC but remains in the nuclei of JAM-A ERΔIEC IEC, revealing a failure of Hippo signaling initiation. Graph depicts scoring of YAP localization (nuclear, cytoplasm, and nuclear + cytoplasm). Five fields of view for each condition were scored to generate an average percentage, and three independent experiments were performed (denoted by the dots). Scale bar, 20 μm. (D) JAM-A-deficient primary IEC exhibit decreased Hippo signaling activity. Representative western blots for p-LATS1 S909, LATS1, p-NF2 S518, NF2, p-YAP S127, YAP, and JAM-A in subconfluent 2D colonoids derived from JAM-A ERΔIEC and JAM-A fl/f l mice. Graphs represent digital densitometry for relative density of p-NF2 S518, p-LATS1 S909, and p-YAP S127. JAM-A ERΔIEC colonoids exhibit decreased NF2 and LATS1 activity, resulting in increased YAP activity relative to JAM-A fl/f l controls. Calnexin serves as a loading control. Data are means ± SEM of three independent experiments. ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001: two-tailed Student’s t test.

Article Snippet: Rabbit Anti-p-NF2/Merlin S518 , Cell Signaling Technology , 9163; RRID: AB_2149793.

Techniques: Cell Culture, Immunostaining, Derivative Assay, Staining, Control, Activity Assay, Western Blot, Two Tailed Test

Journal: iScience

Article Title: JAM-A signals through the Hippo pathway to regulate intestinal epithelial proliferation

doi: 10.1016/j.isci.2022.104316

Figure Lengend Snippet:

Article Snippet: Rabbit Anti-p-NF2/Merlin S518 , Cell Signaling Technology , 9163; RRID: AB_2149793.

Techniques: Recombinant, Plasmid Preparation, SYBR Green Assay, DC Protein Assay, Imaging, Derivative Assay, Sequencing, Negative Control, Software, Microscopy, Spectrophotometry