Microcystin lr

Antibodies were conjugated to Affi-Prep Protein A beads (Bio-Rad Laboratories) at a concentration of 0.3 mg/ml. We lysed 1 × 10⁷ cells in 1 ml NP-40 lysis buffer (50 mM HEPES, pH 7.4, 200 mM KCl, 0.3% NP-40, 10% glycerol, 1 mM EGTA, 1 mM MgCl2, 0.5 mM DTT, and 10 µg/ml each of leupeptin, pepstatin, and chymostatin). To preserve phosphorylation, lysis buffer was supplemented with 1 mM NaVO4 and 0.5 µM microcystin LR (Alexis Biochemicals). Corresponding lysates were centrifuged, incubated with protein A beads coupled with preimmune rabbit IgG at 4 °C for 1 h, and then incubated with protein A beads coupled with specific antibodies at 4 °C overnight. The beads were washed 5x with lysis buffer, boiled in Laemmli sample buffer for 3 min and resolved via SDS–polyacrylamide gel electrophoresis (PAGE).

Microcystin-LR was purchased from Alexis Biochemicals (Lausen, Switzerland), while bleomycin was purchased from Nippon Kayaku Co. (Tokyo, Japan). Human TGF-β1, murine macrophage colony-stimulating factor (M-CSF) and IL-4 were purchased from Pepro Tech (Rocky Hill, New Jersey, USA). FITC and LPS was purchased from Sigma-Aldrich (St. Louis, Missouri, USA).