Rabbit anti phospho p44 42 mapk thr202 tyr204

Human recombinant TGF-β1 and AG1478 were from Calbiochem (La Jolla, CA, USA). EGF was kindly gifted by Serono Lab (Madrid, Spain). Human recombinant HB-EGF, human recombinant TGF-α, MβC, filipin III from Streptomyces filipinensis, nystatin and water-soluble cholesterol were from Sigma-Aldrich (St. Louis, MO, USA). The antibodies used were: mouse anti-β-actin (clone AC-15) from Sigma-Aldrich, rabbit anti-phospho-Akt (Ser473) (D9E) XP, rabbit anti-Akt, rabbit anti-phospho-EGFR (Tyr1068) (D7A5) XP, rabbit anti-EGFR, rabbit anti-phospho-p44/42 MAPK (Thr202/Tyr204), rabbit anti-p44/42 MAPK were from Cell Signaling Technology (Beverly, MA, USA), mouse anti-Caveolin-1 from BD Biosciences (Franklin Lakes, NJ, USA), rabbit anti-Ki67 from AbCam (Cambridge, UK), rabbit anti-TACE/ADAM17 (807-823) from Calbiochem, rabbit anti-NFκB p65, rabbit anti-TβRI (H-100, used in immunocytochemistry), rabbit anti-TβRI (R-20, used in western blot) and rabbit anti-TβRII (C-16) from Santa Cruz Biotechnologies (Dallas, TX, USA). Secondary antibodies: Alexa Fluor 488-conjugated anti-rabbit and anti-mouse from Molecular Probes (Eugene, OR, USA) and ECL Mouse IgG, and Rabbit IgG, HRP-Linked antibodies from GE Healthcare (Buckinghamshire, UK). GM1 was detected with horseradish peroxidase-tagged cholera toxin B subunit from Sigma (St. Louis, MO, USA).

Following incubation with ADH or U0126 as described above, cells were washed with PBS and lysed in RIPA buffer on ice for 30 min. Cell proteins were then collected by centrifugation and quantified with a BCA kit (Thermo Fisher, Waltham, USA). Next, 30 μg of protein per sample were separated on 12% SDS-polyacrylamide gels and transferred to polyvinylidene difluoride (PVDF) membrane. The membrane was subsequently blocked with 5% (w/v) skim-milk and probed with appropriate dilutions of the following primary antibodies overnight at 4 °C: rabbit anti-phospho-p44/42 MAPK (Thr202/Tyr204) (Cell Signaling, Massachusetts, USA), rabbit anti-p44/42 MAP kinase (137F5) (Cell Signaling, Massachusetts, USA), and mouse anti-GAPDH (Protein Tech Group, Chicago, USA). Next, membranes were incubated with secondary antibodies, including goat anti-rabbit IgG, HPR-linked antibody (Cell Signaling, Massachusetts, USA), and goat anti-mouse IgG, HPR-linked antibody (Emarbio Science &Technology, Beijing, China), for 1 h at RT. Proteins were subsequently detected using an enhanced chemiluminescence (ECL) detection kit (Merck Millipore, Missouri, USA) and visualized with a chemiluminescent imaging system (ImageQuant Las4000mini, Tokyo, Japan). Finally, the results were analyzed using ImageJ software (National Institutes of Health, Maryland, USA).