MAPK Phosphorylation Levels in Cells by Western-Blot Analysis

To study the effect of peptides on the ERK pathway EGF stimulation and Western-blot analysis were used. HEK-293T cells were plated on 48-well plate and were serum-starved for overnight. Then cells were treated with the Cf-peptide at 20µM for 5 h in 100 μL DMEM and then stimulated by addition of EGF (Sigma, E9644) in 100 ng/mL concentration. Stimulation was terminated at different time points by adding 35 μL 4× SDS loading buffer. Cells were lysed and 10 μL of each sample was subjected to SDS-PAGE. The proteins of the SDS-gel were transferred to PVDF-membrane by Trans-Blot Turbo Transfer System (Bio-Rad) and the membrane was incubated first by phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) rabbit primery antibody (Cell Signaling, #9101) in 1:2,000 dilution and then with goat anti-rabbit HRP conjugated secondary antibody (Cell Signaling, #7074) in 1:5,000, and after stripping the membrane by anti-p44/42 MAPK (ERK1/2) mouse primary antibody (Cell Signaling, #4696) in 1:2,000 and then with goat anti-mouse HRP conjugated secondary antibody (Merck Milipore, 401215) in 1:10,000. Immobilon^® ECL Ultra Western HRP Substrate reagent (Millipor, WBK L S0500) and Fluorchem FC2 gel documentation system (Alpha Innotech) were used for developing the membrane. The intensities of the protein bands were determined by densitometry and monitored after the stimulation in time. Later the effect of the most effective peptide (bicyclic peptide 16) on the ERK and p38 pathway was examined by Western-blot. In this case HEKT cells were treated with several concentrations of the peptide and then were stimulated by 50 or 200 ng/ml EGF for studying ERK pathway, and 0.4 M Sorbitol for the p38-pathway. Sorbitol induces osmotic stress to the cells which generates activated p38-level (pp-p38), since p38 protein is one of the stress-sensor of the cell. After 10 min cells were lysed and subjected to SDS-PAGE and then subjected to Western-blot as earlier described. Activated (i.e., phosphorylated) ERK and p38 were detected by phospho-p44/42 MAPK (ERK1/2) (see above) and phospho-p38 MAPK (Thr180/Tyr182) rabbit primary antibody (Cell Signaling, #9215) in 1:2000 dilution, respectively. The total ERK and p38 content of the samples were determined by anti-p44/42 MAPK (ERK1/2) mouse primary antibody and anti-p38α MAPK mouse mAb (Cell Signaling, #9228), respectively. Anti-alfa-tubulin mouse mAb antibody (Sigma, T6199) in 1:10,000 was used for loading control. As secondary antibody anti-rabbit antibody (IRDye^® 800CW Goat anti-Rabbit IgG, #926-32211) in 1:5,000 and secondary anti-mouse antibody (IRDye^® 680CW Goat anti-Mouse IgG, #926-68070 in 1: 10,000 were used. Protein bands were detected and quantified with LI-COR Odyssey^® Clx infrared imaging system. Statistical analysis was done using two-sided independent Student’s t-test.