anti talin1 8d4  (Novus Biologicals)

Journal: iScience

Article Title: Rap1 organizes lymphocyte front-back polarity via RhoA signaling and talin1

doi: 10.1016/j.isci.2023.107292

Figure Lengend Snippet: Talin1 is involved in cell polarity downstream of Rap1 (A) Changes of cell elongation of naive WT and Tln1 −/− T cells after stimulation. T cells suspended in RPMI1640 containing 1% BSA were stimulated with CCL21 (100 nM) and examined for cell elongation by imaging cytometry. The data shows the average percentage ±SD (n = 3, a representative of two experiments). (B) Changes in the cell polarization of naive WT and Tln1 −/− T cells as in (A). (C) Chemotactic assays of WT, Tln1 −/− (left panel) and Rap1a −/− Rap1b −/− (right panel) T cells in response to 30 nM CCL21 (n = 3). (D) The amount of RhoA-GTP of Tln1 −/− T cells at 2 min after stimulation with CCL21 (100 nM) measured by flow cytometry. Bars indicate the average MFI of RhoA-GTP ±SD (n = 3, a representative of two experiments). (E) The amount of pMLC of Tln1 −/− T cells at 2 min after stimulation with CCL21 (100 nM). The relative amount of pMLC in mutant T cells was calculated as MLC levels by MFI normalized to the average of MFI of unstimulated WT T cells. (n = 3, a representative of two independent experiments). (F) The clustering of pMLC in WT and Tln1 −/− T cells at 2 min after stimulation with CCL21 (100 nM) was measured by imaging cytometry. Bars indicate the average ±SD (n = 3, a representative of two independent experiments). (G) A representation of confocal images of F-actin, talin1, and pMLC in HT-talin1 knock-in ( Tln1 HT/WT ) T cells stained with HaloTag SaraFluor 650T Ligand. WT T cell ( Tln1 w/w ) was used for negative control of HT-specific staining. Scale bar, 5 μm. (H) Relative levels of pMLC in WT, Rasa3 −/− Sipa1 −/− , Rasa3 −/− Sipa1 −/− Tln1 −/− , and Tln1 −/− T cells were measured by flow cytometry. Bars indicate average ±SD (n = 3, a representative of two independent experiments). (I) The clustering of pMLC (average ±SD) as in (H). (J) The cell elongation (average ±SD) as in (H). Statistical significance for the above data was determined by two-tailed Student’s t test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001.

Article Snippet: Anti-talin1/2 (clone 8D4) and tubulin alpha mAbs were from Sigma Aldrich.

Techniques: Imaging, Cytometry, Flow Cytometry, Mutagenesis, Knock-In, Staining, Negative Control, Two Tailed Test

Journal: iScience

Article Title: Rap1 organizes lymphocyte front-back polarity via RhoA signaling and talin1

doi: 10.1016/j.isci.2023.107292

Figure Lengend Snippet: Localization of talin1 during T cell migration (A) Timelapse imaging of HT-talin1 during T cell migration on immobilized ICAM1 (5 μg/mL) and CCL21 (100 nM). Upper and lower images represent xy and xz projection images, respectively. Dotted lines represent the contact surface. (B) Representative xy and xz projection images of HT-talin1, F-actin, and myosin II in WT and Tln1 HT/wt T cells migrated on immobilized ICAM1 (5 μg/mL) and CCL21 (100 nM). Scale bar, 5 μm. Dotted lines represent the contact surface. (C and D) Super-resolution radial fluctuation (SRRF) image of HT-talin1, F-actin, and myosin II at contact surface (C) and in uropod (D) with enlarged views of dotted areas (right). Arrowheads indicate HT-talin1 colocalized with F-actin (C) and F-actin in the vicinity of myosin II (D). Scale bar, 5 μm. (E) A schematic view of Rap1/talin1 pathways in migrating cells on ICAM-1. Rap1 and talin1 to orchestrate chemotactic migration via both integrin-dependent adhesion and integrin-independent cell polarity. The DATA above are a representative of 2 experiments.

Article Snippet: Anti-talin1/2 (clone 8D4) and tubulin alpha mAbs were from Sigma Aldrich.

Techniques: Migration, Imaging

Journal: iScience

Article Title: Rap1 organizes lymphocyte front-back polarity via RhoA signaling and talin1

doi: 10.1016/j.isci.2023.107292

Figure Lengend Snippet:

Article Snippet: Anti-talin1/2 (clone 8D4) and tubulin alpha mAbs were from Sigma Aldrich.

Techniques: Purification, Transduction, Recombinant, Cell Isolation, Western Blot, Mutagenesis, Software