The cytomegalovirus (CMV) myc-CREB plasmid was created by B. Lonze (Ginty Lab). msMBD3, ratRBBP4, and ratRBBP7 were subcloned into the CMV-myc expression vector. Mutagenesis to create RBBP7, RBBP4 and MBD3 cystine-to-serine mutants, MBD3 lysine-to-alanine mutants, and mycRBBP7WT and mycRBBP7C166S siRNA-resistant plasmids was carried out using a QuikChange multisite-directed mutagenesis kit (Agilent). HA-tagged ratRBBP7 and HA-tagged msHDAC2 were each cloned into AdEasy pShuttle-CMV vectors, and cysteine and lysine mutants were created using QuikChange multisite-directed mutagenesis kit (Agilent). Flag-mCHD4, hCHD5, and hCHD3 were generated as previously described (21 (link)). siRNAs were generated by Invitrogen. The following sequences were used: siRBBP7, 5′-CCACAUAAUGAAACUAUUCUGGCUU-3′ (forward) and 5′-AAGCCAGAAUAGUUUCAUUAUGUGG-3′ (reverse); siRBBP4, 5′-AAAUCUUUCCCUUCAGGCCUGGUCA-3′ (forward) and 5′-UGACCAGGCCUGAAGGGAAA-GAUUU-3′ (reverse).
Protocol detail
Generating Cysteine and Lysine Mutants
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Alanine
Cysteine
Cystine
Cytomegalovirus
Lysine
Mbd3
Mutagenesis
Plasmids
Rbbp7
Serine
Sirnas
Vectors
Corresponding Organization : Medical Research Council
Other organizations : Magna Graecia University
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Variable analysis
independent variables
- Mutagenesis to create RBBP7, RBBP4 and MBD3 cystine-to-serine mutants
- Mutagenesis to create MBD3 lysine-to-alanine mutants
- Creation of mycRBBP7^WT^ and mycRBBP7^C166S^ siRNA-resistant plasmids
- Not explicitly mentioned
- Not explicitly mentioned
- HA-tagged ratRBBP7
- HA-tagged msHDAC2
- Not explicitly mentioned
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