Antibodies against phosphorylated erk1 2

HMVECs were stimulated with Gal-9 M (27.9 nmol/L) for various time points. To determine the role of signaling molecules, HMVECs were incubated with chemical signaling inhibitors for 1 h prior to stimulation with Gal-9 M. To ensure equal loading, protein concentrations of samples were determined using the Pierce bicinchoninic acid protein assay kit (Thermo Fisher Scientific, Rockford, IL, USA). Samples were then analyzed by Western blotting using antibodies against phosphorylated Erk1/2, p38, and Jnk (Cell Signaling Technology, Danvers, MA, USA) [12 (link), 16 (link), 17 (link)]. Immunoblots were stripped and reprobed with β-actin and antibodies to nonphosphorylated proteins to further ensure equal loading.

Rabbit polyclonal polyclonal antibodies recognizing pan-PIPKIγ or Y639-phosphorylated PIPKIγ were generated as described previously [5 (link)]. HA antibody is from Millipore. Antibodies against phosphorylated ERK1/2, total ERK1/2, phosphorylated Akt, total Akt, phosphorylated STAT3, total STAT3 are from Cell Signaling. β-actin and talin antibodies are from Sigma. Phospho-tyrosine antibody is from BD Biosciences. Alexa Flour 555-conjugated phalloidin, Alexa Flour 488-conjugated goat anti-mouse antibody, Alexa Flour 555-conjugated goat anti-rabbit antibody and Alexa Fluor 555-conjugated goat anti-rat antibody are from Molecular probes.

Rabbit polyclonal polyclonal antibodies recognizing pan-PIPKIγ or Y639-phosphorylated PIPKIγ were generated as described previously [5 (link)]. HA antibody is from Millipore. Antibodies against phosphorylated ERK1/2, total ERK1/2, phosphorylated Akt, total Akt, phosphorylated STAT3, total STAT3 are from Cell Signaling. β-actin and talin antibodies are from Sigma. Phospho-tyrosine antibody is from BD Biosciences. Alexa Flour 555-conjugated phalloidin, Alexa Flour 488-conjugated goat anti-mouse antibody, Alexa Flour 555-conjugated goat anti-rabbit antibody and Alexa Fluor 555-conjugated goat anti-rat antibody are from Molecular probes.