Western Blot Analysis of Signaling Proteins

The H358-P and H358-S cells were lysed using modified RIPA lysis buffer (Merck Millipore, Billerica, MA,) containing protease inhibitors (Roche, Indianapolis, IN,) and phosphatase inhibitors (Thermo Scientific, Bremen, Germany). Western blot analysis was performed as described previously using 30 μg protein lysates [79 (link), 80 (link)]. Briefly, lysate were resolved and transferred on nitrocellulose membranes, which was further hybridized with primary antibodies and developed using Luminol reagent (Santa Cruz Biotechnology, Dallas, TX,). Anti-EGFR, phospho-EGFR (Y1068 and Y1197), anti-FAK, phospho-FAK (Y576/577), anti-SRC, and phospho-SRC (Y416) antibodies were obtained from Cell Signaling Technology (Cell Signaling Technology, Beverly, MA). Anti-FRK antibody was obtained from Santa Cruz Biotechnology. Beta-actin antibody was obtained from Sigma (St. Louis, MO). The FAK inhibitor PF-562271 was purchased from Santa Cruz Biotechnology (Dallas, TX). Erlotinib and Dasatinib were purchased from Selleckchem (Houston, TX). DMSO was used as vehicle in all experiments.

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Solanki H.S., Raja R., Zhavoronkov A., Ozerov I.V., Artemov A.V., Advani J., Radhakrishnan A., Babu N., Puttamallesh V.N., Syed N., Nanjappa V., Subbannayya T., Sahasrabuddhe N.A., Patil A.H., Prasad T.S., Gaykalova D., Chang X., Sathyendran R., Mathur P.P., Rangarajan A., Sidransky D., Pandey A., Izumchenko E., Gowda H, & Chatterjee A. (2018). Targeting focal adhesion kinase overcomes erlotinib resistance in smoke induced lung cancer by altering phosphorylation of epidermal growth factor receptor. Oncoscience, 5(1-2), 21-38.

Publication 2018

modified RIPA lysis buffer protease inhibitors phosphatase inhibitors Luminol reagent phospho-SRC (Y416) Beta-actin antibody Erlotinib Dasatinib

Anti antibody Antibodies Antibody Beta actin Buffer Cells Dasatinib Dmso Egfr Erlotinib Inhibitors Luminol Membranes Nitrocellulose Phosphatase Protease inhibitors Protein Ripa Western blot analysis

Corresponding Organization :

Other organizations : Institute of Bioinformatics, KIIT University, Johns Hopkins University, Manipal Academy of Higher Education, Amrita Vishwa Vidyapeetham, Yenepoya University, National Institute of Mental Health and Neurosciences, Johns Hopkins Medicine, Indian Institute of Science Bangalore

Top 5 similar protocols

Variable analysis

independent variables

FAK inhibitor PF-562271
Erlotinib
Dasatinib

dependent variables

EGFR expression
Phosphorylation of EGFR (Y1068 and Y1197)
FAK expression
Phosphorylation of FAK (Y576/577)
SRC expression
Phosphorylation of SRC (Y416)
FRK expression

control variables

DMSO (used as vehicle)

controls

Beta-actin (used as a loading control)

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