Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Focal Adhesion Kinase–Related Nonkinase Inhibits Vascular Smooth Muscle Cell Invasion by Focal Adhesion Targeting, Tyrosine 168 Phosphorylation, and Competition for p130 Cas Binding

doi: 10.1161/atvbaha.111.235549

Figure Lengend Snippet: Figure 3. Focal adhesion kinase–related nonkinase (FRNK) tyrosine phosphorylation at Tyr168 is critical for FRNK inhibition of vascular smooth muscle cell (VSMC) invasion. Rat aortic smooth muscle cells (RASMCs) were infected (100 MOI, 24 hours) with adenovirus (Adv)-expressing green fluorescent pro- tein (GFP)–tagged, wild-type (wt), Tyr168Phe-, Tyr232Phe-, or Tyr168,232Phe-FRNK. A, Average kFRAP (where FRAP indicates fluorescence recovery after photobleaching) values (second1) for peripheral focal adhesions of 20 cells from each of 4 differ- ent experiments (a total of 80 individual total internal reflection fluorescence [TIRF]-FRAP recordings) were compared. Data are meanSEM; *P0.05 vs wtFRNK. B and C, Equal numbers of Adv-infected cells were suspended in serum-free medium and placed in the upper chamber of Matrigel-coated Boyden cham- bers. Angiotensin II (Ang II) (100 nmol/L) was placed in the lower chamber. Cells were allowed to migrate for 2 hours, and cell invasion was quantified as in Figure 2. B, Representative object scoring maps from a single experiment. C, Quantitative analysis of 20 object scoring maps per group from 3 separate experi- ments was compared. Data are meanSEM; *P0.05 vs GFP.

Article Snippet: TIRF- illumination was performed by a 445nm diode laser and/or multiline 300mW Argon laser via a Nikon TIRF II illuminator coupled with a custom beam combiner launch.

Techniques: Phospho-proteomics, Inhibition, Infection, Expressing

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Focal Adhesion Kinase–Related Nonkinase Inhibits Vascular Smooth Muscle Cell Invasion by Focal Adhesion Targeting, Tyrosine 168 Phosphorylation, and Competition for p130 Cas Binding

doi: 10.1161/atvbaha.111.235549

Figure Lengend Snippet: Figure 4. Mutation of Tyr168 enhanced FAT and paxillin interaction. Rat aortic smooth muscle cells (RASMCs) were infected (100 MOI, 24 hours) with adenovirus (Adv) expressing green fluorescent protein (GFP)–tagged wild-type (wt) or Tyr168Phe–focal adhesion kinase– related nonkinase (FRNK). Paired cultures were stimulated with angiotensin II (Ang II) (100 nmol/L, 15–45 minutes). A, Average kFRAP (where FRAP indicates fluorescence recovery after photobleaching) values (second1) for the 80 individual total internal reflection fluo- rescence (TIRF)-FRAP recordings were compared. B, Two-compartment analysis of wtFRNK and Tyr168Phe-FRNK binding kinetics. M1 and M2 represent the fraction of total fluorescence in the “fast” and “slow” compartments, respectively. C and D, RASMCs were infected (100 MOI, 24 hours) with Adv-GFP, Adv-GFP-wtFRNK, or Adv-GFP-Tyr168Phe-FRNK. C, Cell extracts (500 g total protein) were coimmunoprecipitated with anti-paxillin monoclonal antibody (mAb), and immunoblots were probed with anti-GFP mAb (to detect GFP-wtFRNK and GFP-Tyr168Phe-FRNK). The position of molecular weight markers is indicated to the left of each blot. IP indicates immunoprecipitation; WB, Western blot. D, Quantitative analysis of GFP-wtFRNK and GFP-Tyr168Phe-FRNK coimmunoprecipitated with paxillin. Data are meanSEM; n4 experiments. *P0.05 vs wtFRNK.

Article Snippet: TIRF- illumination was performed by a 445nm diode laser and/or multiline 300mW Argon laser via a Nikon TIRF II illuminator coupled with a custom beam combiner launch.

Techniques: Mutagenesis, Infection, Expressing, Binding Assay, Western Blot, Molecular Weight, Immunoprecipitation

Journal: bioRxiv

Article Title: Functionally selective dopamine D1 receptor endocytosis and signaling by catechol and non-catechol agonists

doi: 10.1101/2024.04.15.589637

Figure Lengend Snippet: HEK293 cells were transfected with HA-D1R and β-arrestin2-GFP. The cells were then treated with 3 µM of the indicated agonist and fixed after 10 minutes. The D1R was then detected using antibodies against the HA tag and visualized with alexa594 secondary antibodies. TIRF microscopy was used to image a thin slice of the cells at the plasma membrane. Punctate GFP signal indicates β-arrestin recruitment to the plasma membrane. A) Vehicle treatment did not recruit β-arrestin to the plasma membrane. B) SKF-81297 strongly recruited β-arrestin to the plasma membrane. β-arrestin colocalized in puncta with HA-D1R. C) A-77636 treatment induced β-arrestin recruitment to the plasma membrane that colocalized with the HA-D1R. D) PF-1119 weakly recruited β-arrestin to the plasma membrane. E) Cmpd 41 moderately recruited β-arrestin to the plasma membrane. F) Cmpd 19 strongly recruited β-arrestin to the plasma membrane and colocalized with HA-D1R. Similar results observed in >21 cells across three independent experiments. White boxes indicate the area that is enlarged in the corner of merged images. Scale bar = 10 μm.

Article Snippet: TIRF microscopy was performed using a Nikon Eclipse Ti2 dual epi fluorescence and confocal microscope outfitted with a TIRF imaging apparatus.

Techniques: Transfection, Microscopy, Clinical Proteomics, Membrane

Journal: Cell Death & Disease

Article Title: DOCK8 gene mutation alters cell subsets, BCR signaling, and cell metabolism in B cells

doi: 10.1038/s41419-024-07180-w

Figure Lengend Snippet: Purified splenic B cells from WT and DOCK8 Mut mice were activated with AF647-Fab-goat-anti-mouse Ig (M + G) (10 μg/ml) for 3 min, 5 min, and 7 min, then fixed, permeabilized, and stained for markers following. A , C Representative total internal reflection fluorescence microscopy (TIRFm) images of pSYK and pSHIP1 (100× objective, scale bar = 5 μm). B , D MFI of pSYK and pSHIP-1 was performed using NIS-Elements AR 5.0.1 software. Data shown in the figures are representative of at least three independent experiments. All data are presented as mean ± SEM (* P < 0.05, ** P < 0.01, *** P < 0.001; two-tailed unpaired t-test).

Article Snippet: Visualization was accomplished through Nikon TIRFm microscopy, followed by subsequent image processing with the NIS-elements AR 5.01 software to assess both the MFI.

Techniques: Purification, Staining, Fluorescence, Microscopy, Software, Two Tailed Test

Journal: Cell Death & Disease

Article Title: DOCK8 gene mutation alters cell subsets, BCR signaling, and cell metabolism in B cells

doi: 10.1038/s41419-024-07180-w

Figure Lengend Snippet: A Representative confocal microscopy (CFm) images of phosphorylated WASP (pWASP), F-actin, and BCR (60× objective, scale bar = 2.5 μm). B , C Colocalization of pWASP and F-actin with BCR was performed using NIS-Elements AR 5.0.1 software. D Representative total internal reflection fluorescence microscopy (TIRFm) images of pWASP and F-actin at 3 min, 5 min, and 7 min of activation (100× objective, scale bar = 5 μm). E, F Quantification of MFI for pWASP and F-actin was performed using NIS-Elements AR 5.0.1 software. G, H Quantification of interaction between B cells and mAg in the contact zone, MFI of BCR was performed using NIS-Elements AR 5.0.1 software. J, K Splenic B cells were pre-incubated with B220 and then stimulated with sAg for 0, 5, 10, and 30 min. After fixation and permeabilization, cells were stained with pWASP and F-actin and then detected by flow cytometry. The MFI of pWASP and F-actin in B220 + cells was quantified by FlowJo 10.8.1 software. L The ability of B cells to internalize antigens in WT and Mut mice. I Changes in pWASP expression seen for splenic B cells stimulated with biotin-conjugated F(ab′) 2 anti-mouse Ig(M + G) plus streptavidin for 0 and 5 min by Western blotting. Loading control was total protein and β-Actin. Data shown in the figures are representative of at least three independent experiments. All data are presented as mean ± SEM (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001; two-tailed unpaired t-test).

Article Snippet: Visualization was accomplished through Nikon TIRFm microscopy, followed by subsequent image processing with the NIS-elements AR 5.01 software to assess both the MFI.

Techniques: Confocal Microscopy, Software, Fluorescence, Microscopy, Activation Assay, Incubation, Staining, Flow Cytometry, Expressing, Western Blot, Control, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: Integrins Influence the Size and Dynamics of Signaling Microclusters in a Pyk2-dependent Manner *

doi: 10.1074/jbc.M114.614719

Figure Lengend Snippet: Effect of integrin receptor ligation by ICAM-1 on CD16.NK-92 adhesion and cytolytic granule release. CD16.NK-92 cells were exposed to bilayers containing anti-CD16 antibody (50 mol/μm2) in the presence or absence of ICAM-1. A, percent of CD16.NK-92 cells that formed contacts with the bilayers (empty bars) and percent of the cells that released granules (slashed bars) are shown. The contact formation was determined as apparent change in the cell shape in transmitted light and by accumulation of fluorescent anti-CD16 antibodies at the cell/bilayer interface by TIRF microscopy. Granule release was detected by the appearance of CD107a molecules at the cell/bilayer interface by TIRF microscopy. The data are pooled average results from 3 independent experiments with 10 observation fields in each and are shown as mean ± S.D.; statistically significant differences were observed for both adherent and degranulating cells with p < 0.0001 by two-tailed Student's t test. B, representative images of CD16.NK-92 cells adhered to anti-CD16-containing bilayers with (left panel) or without (right panel) ICAM-1 molecules. Transmitted light images are overlaid with IRM images of the same cell. Dark areas correspond to tight contact between the cells and bilayers. C, average areas of tight contact formed by CD16.NK-92 cells exposed to the bilayers with (slashed bars) or without (empty bars) ICAM-1 are shown. Regions were drawn around tight adhesion areas on the IRM images, and areas of those regions were determined. Representative results of three independent experiments with at least 20 cells analyzed in each group; the data are shown as mean ± S.D., p < 0.0001 by two-tailed Student's t test.

Article Snippet: Images of CD16 clusters bound to fluorescent anti-CD16 antibodies were acquired with a TIRF microscope (Andor Revolution XD system equipped with Nikon TIRF-E illuminator and 100×/1.49 NA objective, Yokogawa CSU-X1 spinning disk confocal, and Andor iXon X3 EM-CCD camera).

Techniques: Ligation, Microscopy, Two Tailed Test