phospho fak  (Cell Signaling Technology Inc)

Journal: APL Bioengineering

Article Title: Survivin modulates stiffness-induced vascular smooth muscle cell motility

doi: 10.1063/5.0252766

Figure Lengend Snippet: Survivin regulates FAK phosphorylation, actin organization, and stress fiber formation. (a) VMSCs were sparsely seeded on fibronectin-coated soft and stiff hydrogels and treated with either YM155 or DMSO. Cells were stained with DAPI, phospho-FAK (Tyr397) antibody and Alexa Fluor 647-phalloidin. Fluorescence images were acquired using a spinning disk confocal microscope with a 63× oil-immersion objective. The outlined boxes indicate the regions shown in the magnified insets. (b) A sum of slices projection of each cell was generated using Fiji/ImageJ, and FAK pY397 clusters were manually counted. 37 cells (soft-DMSO), 44 cells (stiff-DMSO), 33 cells (stiff-0.1 µM YM155), and 37 cells (stiff-2µM YM155) were analyzed from n = 3 independent biological replicates. * p < 0.05, ** p < 0.01.

Article Snippet: For phospho-FAK clustering assay, the same experimental procedures and conditions were used as described above, except cells were stained with phospho-FAK (Tyr397) antibody (1:25; Cat. No. 3283, Cell Signaling Technology) and Alexa Fluor 647-phalloidin (1:200; Cat. No. A22287, Thermo Fisher Scientific).

Techniques: Phospho-proteomics, Staining, Fluorescence, Microscopy, Generated

Journal: Life science alliance

Article Title: Tbx1 plays a critical role in focal adhesion dynamics through paxillin regulation.

doi: 10.26508/lsa.202403151

Figure Lengend Snippet: Figure 1. FA dynamics are altered in Tbx1-depleted cells. (A) Graphs showing the quantification of FA assembly and/or disassembly rate of Tbx1-depleted cells (Tbx1KD) or control cells (NT). (B) Photographs of frames obtained from time-lapse spinning confocal video microscopy movies. Indicated are some of the specific frames that were analysed to assess the number of forming or disassembling FA (arrows) in the lamellipodium of migrating cells, which were transfected with a construct encoding vinculin–GFP fusion protein to mark FAs. (C) Graph showing the number of unstable FA/cell normalized for total FA (FA remaining for less than 30 min). (D) Total Rac1 activity in collagen-stimulated cells was evaluated by the GST-RBD pull-down assay. The levels of total Rac1 or active Rac1 pulled down by GST-RBD analysed by immunoblotting with anti-Rac1 antibody. GAPDH was used as a loading control. (E) ECM-stimulated cells were analysed for the P-FAK (residues Y397 or Y925) and P-ERK1/2 levels by immunoblotting with specific antibody. GAPDH was used as a loading control. Graphs represent quantitative densitometric analysis from at least three experiments (right panels). N = 3 biological replicates. The scale bar represents 10 μm. *P < 0.05, **P < 0.01.

Article Snippet: Membranes were subsequently incubated for 1 h at RT in TBST buffer (125mM Tris–HCl [pH 8.0], 625 mM NaCl, 0.1% Tween-20) containing 5% BSA and further incubated at 4°C for 16 h with the following primary antibodies: phospho-Pxn (#2541; Cell Signaling), phospho-FAK (Y397) (#8556; Cell Signaling), phospho-FAK (Y925) (#sc-11 766; Santa Cruz), phospho-ERK (p44/42) (#9101; Cell Signaling), Pxn (ab32084; Abcam), FAK (#1688; Santa Cruz), ERK (#9102; Cell Signaling), Tbx1 (ab18530; Abcam), PIP5K1c (ab109192; Abcam), talin (SAB4200694; SigmaAldrich), GAPDH (ab125247; Abcam), lamin B1 (ab133741; Abcam).

Techniques: Control, Microscopy, Transfection, Construct, Activity Assay, Pull Down Assay, Western Blot