Phospho p44 42 map kinase phospho erk 1 2

All primary antibodies against PKC isoforms were developed in rabbits and were shown to react specifically with the given PKC isoforms 9 (link),24 (link),27 (link). Anti-PKCα, β, and ε were from Sigma-Aldrich (St. Louis, MO, USA), whereas anti-PKCδ was from Santa Cruz BioTech (Santa Cruz, CA, USA). Specificities of anti-PKC antibodies were also tested by applying isoform-specific blocking peptides, which blocked the immunostaining in all cases 9 (link). Monoclonal mouse antibody against the intermediate filament protein desmin was from DAKO (Glostrup, Denmark). p44/42 MAP kinase (ERK 1/2) and phospho-p44/42 MAP kinase (phospho-ERK 1/2) antibodies were from Cell Signaling Technology (Beverly, MA, USA). In addition, monoclonal rabbit β-actin antibody (Sigma-Aldrich) was employed as internal control.

Throughout the experiments, the following primary antibodies were used: RasGRP3 and phospho-RasGRP3 (Thr133); p44/42 MAP kinase (ERK 1/2) and phospho-p44/42 MAP kinase (phospho-ERK 1/2); Akt and phospho-Akt (Ser473) antibodies for Western blot experiments were obtained from Cell Signaling Technology (Beverly, MA). Anti-RasGRP3 antibody for immunohystochemistry was purchased from Abcam (Cambridge, UK). Ki67 antibody was obtained from DAKO (Glostrup, Denmark). Actin β, ERα, p-ERα (Ser 118) and HER-2 antibodies were purchased from Sigma-Aldrich (St. Louis, MO). IGF-IR and EGFR antibodies were obtained from Santa Cruz Biotechnology (Dallas, TX). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody was obtained from Novus Biologicals (Cambridge, UK). Appropriate secondary antibodies were purchased from Bio-Rad Laboratories (Hercules, CA).