Western Blot Analysis of Signaling Proteins

Cell lysis, SDS-polyacrylamide gel electrophoresis, protein transfer and western blot were performed similarly as before^{31 (link)}. Dual Color Protein Standard (Bio-Rad, Hercules, CA) was used to determine molecular weight. Antibodies used included rabbit anti-phospho-FAK (Tyr397), (Cell Signaling Technology, Danvers, MA), mouse anti-FAK clone 4.47 (EMD Millipore, Darmstadt, Germany), rabbit anti-FoxM1 (Novus Biologicals, Littleton, CO), rabbit anti-Phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (D13.14.4E) (Cell Signaling Technology), rabbit anti-ERK total (Cell Signaling Technology), rabbit anti-phospho-AKT (Ser473) (D9E) (Cell Signaling Technology), mouse anti-GAPDH (EMD Millipore), rabbit anti-AKT total (Cell signaling Technology), rabbit anti p-Src (Tyr416) (Cell Signaling Technology) and mouse anti-total Src (Cell signaling Technology).

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Grindel B.J., Martinez J.R., Tellman T.V., Harrington D.A., Zafar H., Nakhleh L., Chung L.W, & Farach-Carson M.C. (2018). Matrilysin/MMP-7 Cleavage of Perlecan/HSPG2 Complexed with Semaphorin 3A Supports FAK-Mediated Stromal Invasion by Prostate Cancer Cells. Scientific Reports, 8, 7262.

Publication 2018

Dual Color Protein Standard rabbit anti-phospho-FAK (Tyr397), rabbit anti-phospho-AKT (Ser473) (D9E) mouse anti-GAPDH rabbit anti p-Src (Tyr416)

Antibodies Biologicals Cell Clone Erk1 Gapdh Mouse Novus Protein Rabbit Sds polyacrylamide gel electrophoresis Western blot

Corresponding Organization :

Other organizations : Rice University, The University of Texas Health Science Center at Houston, Cedars-Sinai Medical Center

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Variable analysis

independent variables

None explicitly mentioned

dependent variables

Phosphorylation of FAK (Tyr397)
Expression of FAK
Expression of FoxM1
Phosphorylation of ERK1/2 (Thr202/Tyr204)
Total ERK expression
Phosphorylation of AKT (Ser473)
Total AKT expression
Phosphorylation of Src (Tyr416)
Total Src expression

control variables

Cell lysis, SDS-polyacrylamide gel electrophoresis, protein transfer and western blot were performed similarly as before
Dual Color Protein Standard (Bio-Rad, Hercules, CA) was used to determine molecular weight
GAPDH was used as a loading control

positive controls

None explicitly mentioned

negative controls

None explicitly mentioned

Annotations

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