primary antibodies against phosho-fak y397 (focal adhesion kinase  (Cell Signaling Technology Inc)

Journal: International Journal of Molecular Sciences

Article Title: An In Vitro Analysis of TKI-Based Sequence Therapy in Renal Cell Carcinoma Cell Lines

doi: 10.3390/ijms24065648

Figure Lengend Snippet: Effect of cabozantinib on signal transduction in human RCC treatment-naïve 786-O/WT cells and following long-term treatment with sunitinib in human RCC 786-O/S cell lines. ( a ) Cell lysates (20 µg) obtained from wild-type 786-O/WT and 786-O/S cells treated (+) with 12 µM of cabozantinib (Cbz) or untreated (−) with DMSO 0.1% at four time points were analyzed with an immunoblotting assay with specific antibodies for indicated proteins. GAPDH expression was chosen as the loading control for the samples. The optical density (OD) of the area of each band was quantified with ImageJ 1.52a software and normalized to that of GAPDH. Numbers below each line represent the fold changes of the target proteins, which were obtained by dividing the normalized expression from each lane by the normalized expression of the relative untreated controls (set as 1). ( b ) Kinetics of AKT (S473 and T308) in 786-O/WT and 786-O/S cells following treatment with cabozantinib (+Cbz) and untreated control (CTR) at indicated time points. The optical density (OD) of the area of each band of phosphorylated and total AKT was quantified with ImageJ 1.52a software and normalized to that of GAPDH. Phosphorylation kinesis adjusted for AKT (S473/tot AKT and T308/tot AKT). ( c ) Comparative expression levels (fold change) of proteins P-Src, P-FAK and P-S6K between cabozantinib-treated and untreated samples. The relative protein expression (densitometry normalized to the loading control) of each cabozantinib-treated sample was compared with the corresponding DMSO-treated (control, CTR) samples (set as 1) to assess the fold change between the two experimental conditions. Representative immunoblotting image and relative quantification. Full-length blots of independent duplicates are presented in . The figure was generated with Inkscape (version 1.0.2, RRID: SCR_014479).

Article Snippet: They were incubated further overnight with primary antibodies against PAN-MET, phospho-MET Y1234/5 , PAN-AXL, phospho-AXL Y779 , phospho-ERK1/2 T202/Y204 , PAN-AKT, phosphor-Akt T308 , phosho-Akt S473 , phosphoS6 S235/6 , phosho-FAK Y397 (focal adhesion kinase), phosphor-Src family, GAPDH (all antibodies were purchased from Cell Signaling Technology, NEB, Hitchin, UK) and BCL-2 (BD Bioscience, Franklin Lakes, NJ, USA) under agitation at 4 °C overnight.

Techniques: Transduction, Western Blot, Expressing, Software, Generated

Journal: Journal of the American Heart Association

Article Title: Laminin‐221 Enhances Therapeutic Effects of Human‐Induced Pluripotent Stem Cell–Derived 3‐Dimensional Engineered Cardiac Tissue Transplantation in a Rat Ischemic Cardiomyopathy Model

doi: 10.1161/jaha.119.015841

Figure Lengend Snippet: Figure 3. Laminin -221 improves tolerance to hypoxia of human-induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs). A, Immunostaining images of phosphorylated focal adhesion kinase (FAK) (pFAK; Tyr397) (red), laminin-2 (green), and nuclei (blue). Bar=50 µm. B, Western blotting analysis revealed enhancement of expression of pFAK (n=6). C, Quantitative polymerase chain reaction analysis of antiapoptosis-related gene expression in hiPS-CMs under hypoxic conditions: BCL2 (B-cell leukemia/lymphoma 2) and BCL2L1 (Bcl-2–like protein 1) (n=4). D, Western blotting analysis of downstream signaling related protein of FAK: STAT3 (signal transducer and activator of transcription 3) and phosphorylated STAT3 (pSTAT3; pS727-STAT3) (n=6). E and F, FAK inhibitor 14 causes reductions of tolerance to hypoxia of hiPS-CMs: lactate dehydrogenase production (n=7; E) and cell viability (n=10; F). The horizontal line in the middle of each box indicates the median; the top and bottom borders of the box mark the 75th and 25th percentiles, respectively; and the whiskers mark the 90th and 10th percentiles. DAPI indicates 4’,6-diamidino-2-phenylindole; and n.s., not significant.

Article Snippet: Primary antibodies against focal adhesion kinase (FAK) (1:1000, catalog No. 13009), phosphorylated FAK (Tyr397) (1:1000, catalog No. 8556), GAPDH (1:1000, catalog No. 2118), pS727–signal transducer and activator of transcription 3 (STAT3; 1:1000, catalog No. 9134), STAT3 (1:1000, catalog No. 12640), and α-tubulin (1:1000, catalog No. 2125) were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Derivative Assay, Immunostaining, Western Blot, Expressing, Real-time Polymerase Chain Reaction, Gene Expression

Journal: International Journal of Nanomedicine

Article Title: Delivery of dexamethasone from bioactive nanofiber matrices stimulates odontogenesis of human dental pulp cells through integrin/BMP/mTOR signaling pathways

doi: 10.2147/ijn.s97846

Figure Lengend Snippet: Figure 5 Integrin signaling pathway analyses. Notes: (A) mRNA expressions of integrin subsets (α1, α5, β1, and β3), by RT-PCR. GAPDH was used as the housekeeping gene. (B) Protein expressions of intracellular adhesive molecules (p-Paxillin, p-FAK, and RhoA), by Western blot. β-actin was used as the control. Abbreviations: RT-PCR, reverse transcription polymerase chain reaction; p-FAK, phospho-focal adhesion kinase; BGN, bioactive glass nanoparticle; DEX, dexamethasone; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Article Snippet: 15 o n 12 -S ep -2 01 8 F or p er so na l u se o nl y. Powered by TCPDF (www.tcpdf.org) 1 / 1 International Journal of Nanomedicine 2016:11submit your manuscript | www.dovepress.com Dovepress Dovepress 2560 lim et al blocking solution for 1 hour and were incubated with 1:1,000 dilution primary antibody against phospho-focal adhesion kinase (p-FAK; Santa Cruz Biotechnology Inc., Dallas, TX, USA), p-Paxillin (Santa Cruz Biotechnology Inc.), actin (Santa Cruz Biotechnology Inc.), Rho-A (Cell Signaling Technology, Danvers, MA, USA), p-Smad1/5/8 (Cell Signaling Technology), p-p38 (Cell Signaling Technology), and p-Akt (Cell Signaling Technology).

Techniques: Reverse Transcription Polymerase Chain Reaction, Adhesive, Western Blot, Control, Reverse Transcription, Polymerase Chain Reaction