HCASMCs were extracted in lysis buffer, and lysates were incubated with primary antibodies overnight at 4°C, washed three times in lysis buffer, and captured with Protein A/G Plus agarose beads (Santa Cruz Biotechnology) for 3 h at 4°C.
Cells were collected in the above mentioned lysis buffer, and cell lysates were incubated with the primary antibody overnight followed by 90 min incubation with protein A and G Dynabeads purchased from Invitrogen (Grand Island, NY, USA).
GST-RBD, precoupled to glutathion-agarose-beads in RIPA buffer, was added and the lysates were incubated at 4°C for 30 min.
48 h post-transfection, cells were disrupted in 1.5 ml of RIPA lysis buffer and 400 µg of cell lysates were incubated with poly(I∶C)- or control poly(C -coated agarose beads (Sigma) in RIPA lysis buffer supplemented with proteases inhibitors C -coatedagaroseU/ml of RNAse inhibeads (Promega) for 1 h at 4°C.
Briefly, cells were lysed with RIPA buffer (R078 from Sigma-Aldrich) and lysates were incubated with the anti-HA resin for 1 h at 4 °C on a rotating wheel.
Rac activity in MDCK cells was measured using the G-LISA absorbance based Rac1 activation assay kit (Cytoskeleton Inc .. MDCK cells were lysed in buffer provided by the manufacturer and clarified lysates were incubated in Rac-GTP-binding protein-coated wells.
Lysates were incubated in reaction buffer (25 mM HEPES pH 7.5, 50 mM NaCl, 10% glycerol, 0.05% CHAPS, and 5 mM dithiothreitol) in the presence of 50 μM fluorogenic substrate (Biomol, Germany), specific for by caspase-3 (DEVD-AMC) or caspase-9 (Ac-LEHD-AFC).
Modifications consisted of the following: homogenized lysates were incubated in lysis buffer for 1 h at 30 °C prior to loading on the Kingfisher Flex; isopropanol was used instead of ethanol in Wash 1 buffer; specimens were washed twice with Wash I buffer and twice with Wash II buffer; and the temperature of the initial binding step on the Kingfisher was changed to 30 °C for 22.5 min.
Lysates were incubated with 1 × assay buffer and 50 μ M suc-LLVY-AMC at 40°C for 75 min.
The cell pellets were lysed with 100 μl lysis buffer (BD Pharmingen, Cat. No. 559759) and the lysates were incubated with Ac-DEVD-pNA (substrate for caspase-3).
