Microinjection of C3 Transferase

The concentration of each C3 protein preparation is determined with a Biorad protein assay kit, using BSA as a standard. The relative activities of C3 transferase preparations can be determined by ADP-ribosylation of recombinant Rho protein or by microinjection. For the ribosylation assay, 10 ng of recombinant RhoA protein are incubated for 1 h at 37 °C in 50 1 of ADP-ribosylation buffer (50 mM Tris-HCI, pH 7.4, 50 mM NaCI, 5 mM MgCl2, 0.3 mM GTP, 1 mM DTT, 1 iCi [ PJNAD) with varying amounts of C3 protein, ranging from 1 to 200 ng. Gel electrophoresis sample buffer is added, the samples are boiled, and proteins resolved by SDS-polyacrylamide gel electrophoresis (13.5 %). Protein is first visualized with Coomassie blue, and then the gel is destained with many washes to remove any unincorporated [ PjNAD. ADP-ribosylated rho is visualized by autoradiography. 

Each new preparation of C3 protein is also assayed by microinjection into quiescent serum-starved Swiss 3T3 cells, followed by treatment with lysophosphatidic acid, LPA (lOOng/ml for 15 or 30 min Sigma L7260) to stimulate Rho and stress fibre assembly (see Section 6.6). The lowest concentration of C3 that still blocks the LPA effect provides a measure of the relative activity of different batches of C3. 

We inject Swiss 3T3 cells in three different states confluent, quiescent subconfluent with serum and subconfluent without serum. 

Subconfluent, with serum Cells are plated onto coverslips at a density of 6 x 10 cells in DMEM/10 % PCS and allowed to attach for 2-3 h before microinjection. 

Routinely, 10 x stock solutions of recombinant C3 protein (0.7-1 mg/ ml) are stored at 4 °C. Long term storage of more concentrated stocks (4-5mg/ml) is at -20 °C. Appropriate dilutions of C3 transferase for microinjection are made up fresh on the day of injection in 150mM NaCI, 50 mM Tris pH 7.5, 5mM MgCb, on ice. So as to be able to identify injected cells later, C3 protein is co-injected with either rat immunoglobulin (final concentration 0.5mg/ml [Pierce cat. no. 31885]) or PITC- or Texas Red-lysinated dextrans (final concentration 2mg/ml [Molecular Probes D1820 and D1863]). This mixture is centri- 

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