Journal: Journal of Medicinal Chemistry

Article Title: Switching Roles—Exploring Concentration-Dependent Agonistic versus Antagonistic Behavior of Integrin Ligands

doi: 10.1021/acs.jmedchem.4c02111

Figure Lengend Snippet: Effects of ligands on cellular expression and activation of integrin-related signaling molecules FAK and p44/42 (erk-1/2) . Integrin αvβ3+ M21 cells, α5β1+ OV-MZ-6, or αvβ6+ HN cells were incubated with 1 μM of L1 (αvβ3), cilengitide (L2; αvβ3/αvβ5), L3 (αvβ6), or L5 (α5β1) for 24 h, and the cellular expression and activation levels of FAK and p44/42 (erk-1/2) were detected by Western blot analysis. Signal intensities for FAK and p-FAK, respectively, were evaluated by use of the Bio-Rad Imager Gel Doc XR+ and a ChemiDoc XRS+ Systems and the software Image Lab. Shown are typical representative Western blot images and the corresponding histograms depicting GAPDH-normalized signal intensities as n -fold by setting the values obtained for untreated cells to “1” (drawn as black line). For each cell line, cutouts of identical gels are depicted.

Article Snippet: For Western blot analysis, the following antibodies were used: (p-)p44/42 (erk-1/2) (1) 1:300 dilution of rabbit-anti-human p44/42 (erk-1/2) antibody (Cell Signaling Technology, Frankfurt, Germany, #9102) in 3% (w/v) bovine serum albumin (BSA)/TBS-T, (2) 1:5,000 dilution goat-anti-rabbit horseradish-conjugated secondary antibody in 1% (w/v) BSA/TBS-T. Phospho-p44/42 (erk-1/2) : (1) 1:200 dilution rabbit-anti-human p44/42 (erk-1/2) (Thr202/Tyr204) antibody (Cell Signaling Technology, #9101) in 3% (w/v) BSA/TBS-T, (2) 1:5,000 dilution goat-anti-rabbit horseradish-conjugated secondary antibody in 1% (w/v) BSA/TBS-T. FAK: (1) 1:300 dilution mouse-anti-human FAK antibody (Cell Signaling Technology, #9102) in 3% (w/v) BSA/TBS-T, (2) 1:5,000 dilution goat-anti-mouse horseradish-conjugated secondary antibody in 1% (w/v) BSA/TBS-T. Phospho-FAK: (1) 1:300 dilution mouse-anti-human p-FAK antibody (Cell Signaling Technology) in 3% (w/v) BSA/TBS-T, (2) 1:5,000 dilution goat-antimouse horseradish-conjugated secondary antibody in 1% (w/v) BSA/TBS-T.

Techniques: Expressing, Activation Assay, Incubation, Western Blot, Software