Generation of Ack1 Antibodies

Polyclonal antibody against Ack1 (384) was generated using the GST fusion proteins corresponding to the Pro-rich region (amino acids 502–1008), as described elsewhere [29 (link)]. The monoclonal antibody against Ack1 was generated by Leitat (Barcelona, Spain) and its efficiency has been described previously [18 (link)]. As antigen, we used the same GST fusion protein encoding amino acids 502–1008 of Ack1 as for polyclonal antibodies. Mice were injected with the purified protein, and standard procedures were followed to obtain the antibodies [59 ]. The monoclonal antibody against CAMKII-α was from Affinity Bioreagents (Golden, CO). The goat anti-rabbit-HRP secondary antibody used in the Western Blot analysis was from Sigma (St. Louis, MO), and the antibody against mouse True Blot Ultra WAS from eBioscience (San Diego, CA). Alexa Fluor-488 goat anti-rabbit immunoglobulin G (IgG) (H + L) and Alexa Fluor-546 goat anti-mouse immunoglobulin G (IgG) (H + L) were supplied by Invitrogen (Carlsbad, CA). Protein G-sepharose beads were from Sigma-Aldrich (St. Louis, MO).