The PH domains of PLCδ1 (1–170), Bruton's tyrosine kinase (1–177), Akt protein kinase (1–167), and dynamin (508–652) were amplified with the Advantage Klentaq polymerase mix (CLONTECH Labs, Inc., Palo Alto, CA) from human cDNAs (marathon cDNA from brain and K562 leukemia cells; CLONTECH Labs, Inc.) with the following primer pairs:
PLCδ: 5′-GGCATGGACTCGGGCCGGGACTTCCTG-3′, 5′-AAGATCTTCCGGGCATAGCTGTCG-3′;
Btk: 5′-CCAAGTCCTGGCATCTCAATGCATCTG-3′,
5′-TGGAGACTGGTGCTGCTGCTGGCTC-3′;
Akt: 5′-GTCAGCTGGTGCATCAGAGGCTGTG-3′,
5′-CACCAGGATCACCTTGCCGAAAGTGCC-3′;
Dyn: 5′-ATGCTCAGCAGAGGAGCAACCAGATG-3′,
5′-GAGTCCACAAGATTCCGGATGGTCTC-3′.
The amplified products were subcloned into the PGEM-Easy T/A cloning vector (Promega Corp. , Madison, WI) and sequenced with dideoxy sequencing (thermosequenase; Amersham Corp. ). A second amplification reaction was performed from these plasmids with nested primers that contained restriction sites for appropriate cloning into the pEGFP-N1 (PLCδ, Btk, and Akt) or pEGFP-C1 (dynamin) plasmids (CLONTECH Labs, Inc.) to preserve the reading frame. Plasmids were transfected into COS-7 cells or NIH-3T3 cells and cell lysates were resolved by SDS-PAGE followed by Western blot analysis for the presence of the GFP fusion proteins using a polyclonal antibody against GFP (CLONTECH Labs, Inc.).
Mutations were created in the PHPLCδ–GFP fusion plasmid by the QuickChange™ mutagenesis kit (Stratagene, La Jolla, CA). For practical purposes, a SalI site was introduced into the PH domain sequence which changed S34 to a T but this substitution did not change any characteristic compared with the wild-type protein. All mutations were confirmed by dideoxy sequencing and the expression of the fusion protein by Western blot analysis.
PLCδ: 5′-GGCATGGACTCGGGCCGGGACTTCCTG-3′, 5′-AAGATCTTCCGGGCATAGCTGTCG-3′;
Btk: 5′-CCAAGTCCTGGCATCTCAATGCATCTG-3′,
5′-TGGAGACTGGTGCTGCTGCTGGCTC-3′;
Akt: 5′-GTCAGCTGGTGCATCAGAGGCTGTG-3′,
5′-CACCAGGATCACCTTGCCGAAAGTGCC-3′;
Dyn: 5′-ATGCTCAGCAGAGGAGCAACCAGATG-3′,
5′-GAGTCCACAAGATTCCGGATGGTCTC-3′.
The amplified products were subcloned into the PGEM-Easy T/A cloning vector (
Mutations were created in the PHPLCδ–GFP fusion plasmid by the QuickChange™ mutagenesis kit (Stratagene, La Jolla, CA). For practical purposes, a SalI site was introduced into the PH domain sequence which changed S34 to a T but this substitution did not change any characteristic compared with the wild-type protein. All mutations were confirmed by dideoxy sequencing and the expression of the fusion protein by Western blot analysis.