![Figure 2. AML1/ETO modulates RARβ2 promoter activity through the βRARE binding site due to its interaction with RARα. (A) Schematic representation of the RARβ2 reporter constructs. The sequences of the region of the −5kb+155bp RARβ2pr-LUC reporter vector15 containing the βRARE and the TATA box (from −59 bp to −23 bp) carrying the wild-type 5′-GGTTCAC-3′ direct motif of the βRARE site (RAREwt)15 or a 5′-aacTCAC-3′ selective mutation at this site (RAREmut) are indicated. (B) Human 293T cells were transiently cotransfected with increasing amounts of the pCDNA3-AML1/ETO expression vector (20 ng and 40 ng) and 1 μg of RARβpr-LUC carrying the RAREwt or the RAREmut site. A cotransfected vector encoding the β-galactosidase (pSV-βgal) was used as an internal control for normalization of the reactions. After transfections the cells were treated (⊡) or not (□) with 1 μM RA for 24 hours. The results represent the average of 3 independent evaluations ± SD. (C) Coimmunoprecipitation and Western blot experiments performed in human U937 WT, U937 stably transfected with an HA-tagged AML1/ETO cDNA (A/E-HA), or with an empty vector (mock), and in human SKNO-1 cells lines. Ip- indicates the antibody used for the coimmunoprecipitation; IgG, rabbit serum used as nonspecific antibody. Coimmunoprecipitates were analyzed by Western blot (WB) with antibodies detecting the expression levels of AML1/ETO (A/E), AML1, and RARα proteins in different samples and in whole cell lysates (input). The difference in size of the AML1/ETO fusion protein between the A/E-HA and SKNO-1 is due to the HA-tagged domain, which increased the molecular weight of the AML1/ETO product in A/E-HA cells. (D) Nuclear extracts were prepared from U937 mock and A/E-HA cells and analyzed (50 μg) by Western blot for the expression of the RARα and AML1/ETO products by using α-RARα and α-HA antibodies, respectively. The expression of β-tubulin confirmed protein loading. RA binding activity was measured in (•) mock and (○) A/E-HA nuclear extracts labeled with 10 nM [3H]-RA in the absence or in the presence of 200-fold molar excess of unlabeled RA (▴, mock; △, A/E-HA) to determine nonspecific binding by HPLC using a Superose 6 HR 10/30 size exclusion column.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/109/10/10.1182_blood-2006-09-045781/4/zh80100700950002.jpeg?Expires=1726820989&Signature=Gzs3IvU5tMRWqnvRn4DyZGKc6aDX-xAC2xPBYefjrLBF-4BDbWVk6FzlmpGJaOzYOxnTAHkSOOptsei2kxTFHhjqAdNBrDNba3SeAhz9~X0rmpG7u2LhuXtTfVA21s3Zfthy3XEix1pk27WrtEwJGDXnxIsHasfvdhWfIIDo7KHpCnfPnAl8bX3Y50beRXgiUEHGxsh5du7OlffU9WAyYi-AyN2SFwE7Kj~j-5S8TFzcyRpklXIdzjYEv13Kb47bhS4hVtxnzXWj4eEveDFc~YNMOv8E2GxSij8EiBBaOhgJGWAjAw2GsW6jjpi8jR5aPKCK63YhJHV6AWKhbmCbZw__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
AML1/ETO modulates RARβ2 promoter activity through the βRARE binding site due to its interaction with RARα. (A) Schematic representation of the RARβ2 reporter constructs. The sequences of the region of the −5kb+155bp RARβ2pr-LUC reporter vector15 containing the βRARE and the TATA box (from −59 bp to −23 bp) carrying the wild-type 5′-GGTTCAC-3′ direct motif of the βRARE site (RAREwt)15 or a 5′-aacTCAC-3′ selective mutation at this site (RAREmut) are indicated. (B) Human 293T cells were transiently cotransfected with increasing amounts of the pCDNA3-AML1/ETO expression vector (20 ng and 40 ng) and 1 μg of RARβpr-LUC carrying the RAREwt or the RAREmut site. A cotransfected vector encoding the β-galactosidase (pSV-βgal) was used as an internal control for normalization of the reactions. After transfections the cells were treated (⊡) or not (□) with 1 μM RA for 24 hours. The results represent the average of 3 independent evaluations ± SD. (C) Coimmunoprecipitation and Western blot experiments performed in human U937 WT, U937 stably transfected with an HA-tagged AML1/ETO cDNA (A/E-HA), or with an empty vector (mock), and in human SKNO-1 cells lines. Ip- indicates the antibody used for the coimmunoprecipitation; IgG, rabbit serum used as nonspecific antibody. Coimmunoprecipitates were analyzed by Western blot (WB) with antibodies detecting the expression levels of AML1/ETO (A/E), AML1, and RARα proteins in different samples and in whole cell lysates (input). The difference in size of the AML1/ETO fusion protein between the A/E-HA and SKNO-1 is due to the HA-tagged domain, which increased the molecular weight of the AML1/ETO product in A/E-HA cells. (D) Nuclear extracts were prepared from U937 mock and A/E-HA cells and analyzed (50 μg) by Western blot for the expression of the RARα and AML1/ETO products by using α-RARα and α-HA antibodies, respectively. The expression of β-tubulin confirmed protein loading. RA binding activity was measured in (•) mock and (○) A/E-HA nuclear extracts labeled with 10 nM [3H]-RA in the absence or in the presence of 200-fold molar excess of unlabeled RA (▴, mock; △, A/E-HA) to determine nonspecific binding by HPLC using a Superose 6 HR 10/30 size exclusion column.
