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

Journal: The FEBS journal

Article Title: Cadherin-6 controls neuronal migration during mouse neocortical development via an integrin-mediated pathway.

doi: 10.1111/febs.70150

Figure Lengend Snippet: Fig. 7. KD and overexpression of Cdh6 did not alter the levels of Paxillin protein and phosphorylated AKT. (A) Immunostaining for Paxillin (top, red) or pFAK (red, bottom) and EGFP (green) using sections from the E18.0 cerebral cortex, which were electroporated with control (n = 3) or Cdh6 KD vector (n = 3) (left) or control (CAG empty) (n = 3) or CAG-Cdh6-HA vectors (n = 3) (right) with CAG-EGFP vectors at E14.0. (B) Western blot analysis of neocortical cells electroporated with CAG empty or CAG-Cdh6- HA vectors using antibodies against HA, Paxillin, pFAK, FAK, and GAPDH (a loading control) (n = 3). Sections in (A) were stained with DAPI (magenta). Scale bar: 50 lm in (A). KD, knockdown.

Article Snippet: The following primary antibodies were used in this study: CDH6 (sheep; R&D Systems, AF2715, 1 : 200), HA (rat; Roche, 11867423001, 1 : 1500), GAPDH (mouse; Santa Cruz Biotechnology, Dallas, TX, USA, sc-32233, 1 : 1000), integrin b1 (rabbit; Abcam, ab183666, 1 : 1200), b-Actin (mouse; Sigma-Aldrich, MO, USA, A5441, 1 : 1000), Paxillin (rabbit; Abcam, ab32084, 1 : 1000), pFAK (Y397) (rabbit; CST, 3283S, 1 : 1500), and FAK (mouse; BD, 610088, 1 : 1000).

Techniques: Over Expression, Immunostaining, Control, Plasmid Preparation, Western Blot, Staining, Knockdown

Journal: iScience

Article Title: Targeting FAK improves the tumor uptake of antibody-drug conjugates to strengthen the anti-cancer responses

doi: 10.1016/j.isci.2024.111536

Figure Lengend Snippet: FAK inhibitor IN10018 effectively eradicates CAFs derived from normal fibroblasts (A) The IF staining figures for NIH/3T3, NIH/3T3 co-cultured with CT26 cells, and NIH/3T3 co-cultured with NCI-N87 cells. The indicated cells were treated with PBS and 3 μm of IN10018 for 5 days. Phospho FAK Y397, FAP, and DAPI signals were recorded by IF staining. (B–C) Western blot results for the IN10018-treated NIH/3T3 co-cultured with CT26 (B) or NCI-N87 (C) cells. The cells were treated with PBS and 3 μm of IN10018 for 3 or 5 days. The protein of NIH/3T3 was harvested for western blot to check the expression of the targets. (D) Clonogenic assay for IN10018-treated NIH/3T3 co-cultured with CT26 cells or NCI-N87 cells. The cells were treated with PBS and 3 μm of IN10018 for 10 days and then the NIH/3T3 cell colonies were stained with crystal violet. (E) The data analysis of colony assay from (D) by ImageJ. Data represent mean ± SEM. The unpaired student’s t test was used for the statistical analysis. NS, non-significant; ∗∗p < 0.01. Scale bar: 40 μm.

Article Snippet: Rabbit polyclonal anti-Phospho FAK Y397 , Thermo Fisher Scientific , Cat#44-624G; RRID: AB_2533701.

Techniques: Derivative Assay, Staining, Cell Culture, Western Blot, Expressing, Clonogenic Assay, Colony Assay

Journal: iScience

Article Title: Targeting FAK improves the tumor uptake of antibody-drug conjugates to strengthen the anti-cancer responses

doi: 10.1016/j.isci.2024.111536

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-Phospho FAK Y397 , Thermo Fisher Scientific , Cat#44-624G; RRID: AB_2533701.

Techniques: Recombinant, Bicinchoninic Acid Protein Assay, TUNEL Assay, Staining, Software