talin 8d4 antibody  (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

Journal: Developmental cell

Article Title: Dynamic Podosome-Like Structures in Nascent Phagosomes Are Coordinated by Phosphoinositides.

doi: 10.1016/j.devcel.2019.05.028

Figure Lengend Snippet: Figure 2. Comparison of Podosomes and Podosome-like Structures Comparison of podosomes formed by resting human macrophages (left) and podosome-like structures formed during frustrated phagocytosis of IgG-opsonized glass (center-left). Stained for active b-2 integrins, talin, vinculin, paxillin, myosin IIa, and ArpC2. Insets show merged images of F-actin (red) and the specified protein (green). Scale bars, 5 mm and 1 mm (insets). Right panels are line scans for F-actin (red) and the specified protein (green) from indicated region highlighted in yellow on corresponding image on left.

Article Snippet: Vinculin (MAB3574) and Arpc2 (07-227) antibodies were from EMDMillipore, myosin IIa nonmuscle (M8064) antibody from Sigma Aldrich, talin (8D4) antibody from Novus Biologicals, active b-2 integrin (MEM-148) antibody from Bio-Rad Laboratories Inc., WASP (sc-13139) from Santa Cruz Biotechnology, and paxillin (610051) antibody from BD Transduction Laboratories.

Techniques: Comparison, Staining

Journal: Journal of Cell Science

Article Title: N-WASP-directed actin polymerization activates Cas phosphorylation and lamellipodium spreading

doi: 10.1242/jcs.134692

Figure Lengend Snippet: Neither actomyosin contractility nor talin-dependent focal adhesion is required for CasSD phosphorylation. (A–D) CasSD phosphorylation during spreading on fibronectin did not decrease with the treatment of blebbistatin (Bb). However, FAK phosphorylation at residue Y397 was significantly impaired (*P<0.05.). Cells were pre-incubated in medium containing DMSO or 50 µM blebbistatin for 30 min and plated on fibronectin-coated dishes for the indicated time before being lysed for western blot assay (IB). sus, suspension. A.U., arbitrary units. (E–H) F-actin staining confirmed that stress fibers were missing in the cells treated with blebbistatin (F,H). The pY165 signal of Cas was comparable in control and treated cells (E,F). FAK phosphorylation at residue Y397 was greatly reduced with loss of focal adhesion structure in blebbistatin-treated cells (G,H). All samples were fixed 20 min after plating. (I–K) Western blots showed comparable CasSD phosphorylation at residue Y165 in talin-deficient cells 10 min after plating on fibronectin although levels of pY165 decreased at later time-points (I). The profile of phosphorylation at residue Y410 was similar to that for residue Y165, except that a smeared signal at a lower molecular mass was observed at later time-points. (J) pY165 Cas∶total Cas and (K) pY410 Cas∶total Cas ratios in different experiments were normalized against control samples (con) at the 60-min time-point. Depletion of talin2 in talin1−/− fibroblasts was examined by total talin antibody 8d4. tl2, talin2. Means±s.d., three repeats. Two-tailed paired Student's t-test was performed. (L) Immunostaining with talin antibody and an antibody against Cas phosphorylated at residue Y165 confirmed that reduction of total talin did not affect CasSD phosphorylation when the cell was still spread. Cells were fixed 15 min after plating. Scale bars: 10 µm.

Article Snippet: Antibodies against the following proteins were used: talin (talin1 and talin2, monoclonal clone 8d4, Sigma), GAPDH (Santa Cruz Biotechnology), p130Cas (raised in goat, Santa Cruz), p130Cas (raised in rabbit, polyclonal, GeneTex), p130Cas (monoclonal, BD Transduction), paxillin (BD Transduction), FAK (which does not recognize FRNK, BD Transduction), Src (mAb327, Calbiochem), phospho-FAK (pY397, Biosource International), FAK (polyclonal recognizing full-length FAK and FRNK, Invitrogen), phospho-FAK (pY861, Invitrogen), phospho-Src (pY416, Cell Signaling), phospho-p130Cas (PY165, Cell Signaling), phospho-p130Cas (pY410, Cell Signaling), integrin β3 (polyclonal, Chemicon), active β1 integrin (clone 9EG7, BD Pharmingen), and N-WASP (affinity-purified, polyclonal, raised in rabbit, a gift provided by Marc W. Kirschner at Harvard Medical School).

Techniques: Incubation, Western Blot, Staining, Two Tailed Test, Immunostaining