Figure 4.
Binding of the Tal1 and SP-1 proteins to the endogenous D1 promoter as assessed by chromatin immunoprecipitation. (A) Physical association between the Sp1 and Tal1 transcription factors. HEK 293 cells were cotransfected with 8 μg mammalian expression vectors for Sp1 and Tal1. Cell lysates were immunoprecipitated with the indicated antibodies or affinity-purified mouse IgG. Immune complexes were analyzed by SDS 6% polyacrylamide gel for Sp1 and by SDS 10 % polyacrylamide gel for Tal1 analysis and sequential immunoblotting with Sp1 and Tal1 antibodies. Total extracts (60 μg) derived from 293 transfected cells were used as input control. (B) Total cell lysates (2 mg) derived from primary thymocytes obtained from the indicated mice (4 weeks of age) were immunoprecipitated with antibodies against either Sp1 (B) or Tal1 (C). Immune complexes were analyzed by immunoblotting with the goat polyclonal antibodies against Sp1 or Tal1. Immune complexes were analyzed by SDS 6% polyacrylamide gel for Sp1 and by SDS 10% polyacrylamide gel for Tal1 analysis. (B, left) Total extract (60 μg) derived from indicated mice was used as input control. (D) Schematic representation of D1 promoter; Sp1 binding sites are indicated. (E) N3-232 T cells were processed for chromatin immunoprecipitation with antibodies against both Sp1 and Tal1. The immunoprecipitates were analyzed by PCR with oligonucleotide primes specific for the indicated region of the mouse cyclin D1 promoter. (F) Primary T cells derived from the indicated mice were processed for chromatin preparation and then subjected to immunoprecipitation with antibodies against either Sp-1 or IgG control antibodies. The immunoprecipitates were analyzed by PCR with oligonucleotide primes specific for the indicated region of the mouse D1 promoter. (G) Primary T cells derived from the indicated mice (4 weeks of age) were processed for chromatin preparation and then subjected to immunoprecipitation with antibodies against either Tal1 or IgG control antibodies. The immunoprecipitates were analyzed by PCR with oligonucleotide primers specific for region 3 of the mouse cyclin D1 promoter.

Binding of the Tal1 and SP-1 proteins to the endogenous D1 promoter as assessed by chromatin immunoprecipitation. (A) Physical association between the Sp1 and Tal1 transcription factors. HEK 293 cells were cotransfected with 8 μg mammalian expression vectors for Sp1 and Tal1. Cell lysates were immunoprecipitated with the indicated antibodies or affinity-purified mouse IgG. Immune complexes were analyzed by SDS 6% polyacrylamide gel for Sp1 and by SDS 10 % polyacrylamide gel for Tal1 analysis and sequential immunoblotting with Sp1 and Tal1 antibodies. Total extracts (60 μg) derived from 293 transfected cells were used as input control. (B) Total cell lysates (2 mg) derived from primary thymocytes obtained from the indicated mice (4 weeks of age) were immunoprecipitated with antibodies against either Sp1 (B) or Tal1 (C). Immune complexes were analyzed by immunoblotting with the goat polyclonal antibodies against Sp1 or Tal1. Immune complexes were analyzed by SDS 6% polyacrylamide gel for Sp1 and by SDS 10% polyacrylamide gel for Tal1 analysis. (B, left) Total extract (60 μg) derived from indicated mice was used as input control. (D) Schematic representation of D1 promoter; Sp1 binding sites are indicated. (E) N3-232 T cells were processed for chromatin immunoprecipitation with antibodies against both Sp1 and Tal1. The immunoprecipitates were analyzed by PCR with oligonucleotide primes specific for the indicated region of the mouse cyclin D1 promoter. (F) Primary T cells derived from the indicated mice were processed for chromatin preparation and then subjected to immunoprecipitation with antibodies against either Sp-1 or IgG control antibodies. The immunoprecipitates were analyzed by PCR with oligonucleotide primes specific for the indicated region of the mouse D1 promoter. (G) Primary T cells derived from the indicated mice (4 weeks of age) were processed for chromatin preparation and then subjected to immunoprecipitation with antibodies against either Tal1 or IgG control antibodies. The immunoprecipitates were analyzed by PCR with oligonucleotide primers specific for region 3 of the mouse cyclin D1 promoter.

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