Heat shock transcription factor (HSF) is a multidomain protein that exists as a monomer under nor... more Heat shock transcription factor (HSF) is a multidomain protein that exists as a monomer under normal conditions and is reversibly induced upon heat shock to a trimeric state that binds to DNA with high affinity. The maintenance of the monomeric state is dependent on hydrophobic heptad repeats located at the amino- and carboxy-terminal regions which have been proposed to form an intramolecular coiled-coil structure. In a systematic deletion analysis to identify other regions of HSF that may be required to regulate its oligomeric state, we have found that local sequences encompassing the carboxy-terminal end of the DNA binding domain and a broad region of HSF between the heptad repeats also contribute to this regulation. Immunocytochemical analysis of mutant HSF proteins revealed a canonical motif required for nuclear localization. HSF proteins lacking the nuclear localization signal remain in the cytoplasm, but these HSFs nonetheless exhibit reversible heat stress-inducible trimeriza...
Hearts were harvested from animals and flash frozen in liquid nitrogen. Tissue was pulverized and... more Hearts were harvested from animals and flash frozen in liquid nitrogen. Tissue was pulverized and homogenized in 25mM HEPES, pH 7.4, 4 mM EDTA, 1.0 mM PMSF and Roche complete protease inhibitor cocktail. The extract was then centrifuged at 8,000-x g for 30 minutes at 4oC. The pellet was then resuspended in 20mM Tris, pH 6.8, 1.0 mM EDTA and 1.0 % SDS and briefly sonicated into solution. Protein concentrations for supernatant and pellet were determined using Bio-Rad protein assay kit. Equal amounts of protein extracts (10-20 μg) were loaded and separated by SDS-PAGE. Proteins were then transferred electrophoretically from the gels to Immobilon-P (Millipore) membrane. Blots were blocked in Tris Buffered Saline-Tween 20 (TBST) containing 5 % (w/v) dry milk followed by incubation for 2 hrs with the respective primary antibody diluted in TBS buffer. Blots were then washed three times for 10 min each in TBST and incubated with anti-rabbit (1:25000) or anti-mouse (1:10000) IgG horseradish ...
Hox genes encode transcriptional regulatory proteins that are largely responsible for establishin... more Hox genes encode transcriptional regulatory proteins that are largely responsible for establishing the body plan of all metazoan organisms. A subset of Hox genes is expressed during the period of organogenesis and into adulthood. hoxb-13 is a recently-described member of the Hox gene family that is expressed in the spinal cord, hindgut, and urogenital sinus during embryogenesis. Northern blot and in situ hybridization analyses of hoxb-13 expression in adult mouse tissues were performed. hoxb-13 mRNA is restricted to the prostate gland and distal colon in adult animals. In situ hybridization of mouse prostate tissue demonstrated that hoxb-13 is expressed in the epithelial cells of the ventral, dorsal, lateral, and anterior prostate lobes. Accumulation of hoxb-13 mRNA is not diminished following castration. These data demonstrate that hoxb-13 expression is androgen-independent in mouse prostate glands. The identification of hoxb-13 as an androgen-independent gene expressed in adult mouse prostate epithelial cells provides a new potential target for developing therapeutics to treat advanced prostate cancer.
Backgrounds and Objectives Small Heat Shock Proteins (HSPs) display chaperone-like activities and... more Backgrounds and Objectives Small Heat Shock Proteins (HSPs) display chaperone-like activities and protect cells against apoptosis and ischemia/reperfusion (I/R) injury. HSPB2 is expressed at high l...
... OF HSF [9] [9] P urificationof Heat Shock T ranscription F actorof Drosophila By MIN ZHONG, J... more ... OF HSF [9] [9] P urificationof Heat Shock T ranscription F actorof Drosophila By MIN ZHONG, JAN WISNIEWSKI, MICHAEL FRITSCH, GAKU MIZUGUCHI, ANDRAS OROSZ, PAUL ... 2 j. Clos, JT Westwood, PB Becker, S. Wilson, K. Lambert, and C. Wu, Cell63, 1085 (1990). ...
Heat shock transcription factor (HSF) is a multidomain protein that exists as a monomer under nor... more Heat shock transcription factor (HSF) is a multidomain protein that exists as a monomer under normal conditions and is reversibly induced upon heat shock to a trimeric state that binds to DNA with high affinity. The maintenance of the monomeric state is dependent on hydrophobic heptad repeats located at the amino- and carboxy-terminal regions which have been proposed to form an intramolecular coiled-coil structure. In a systematic deletion analysis to identify other regions of HSF that may be required to regulate its oligomeric state, we have found that local sequences encompassing the carboxy-terminal end of the DNA binding domain and a broad region of HSF between the heptad repeats also contribute to this regulation. Immunocytochemical analysis of mutant HSF proteins revealed a canonical motif required for nuclear localization. HSF proteins lacking the nuclear localization signal remain in the cytoplasm, but these HSFs nonetheless exhibit reversible heat stress-inducible trimeriza...
Hearts were harvested from animals and flash frozen in liquid nitrogen. Tissue was pulverized and... more Hearts were harvested from animals and flash frozen in liquid nitrogen. Tissue was pulverized and homogenized in 25mM HEPES, pH 7.4, 4 mM EDTA, 1.0 mM PMSF and Roche complete protease inhibitor cocktail. The extract was then centrifuged at 8,000-x g for 30 minutes at 4oC. The pellet was then resuspended in 20mM Tris, pH 6.8, 1.0 mM EDTA and 1.0 % SDS and briefly sonicated into solution. Protein concentrations for supernatant and pellet were determined using Bio-Rad protein assay kit. Equal amounts of protein extracts (10-20 μg) were loaded and separated by SDS-PAGE. Proteins were then transferred electrophoretically from the gels to Immobilon-P (Millipore) membrane. Blots were blocked in Tris Buffered Saline-Tween 20 (TBST) containing 5 % (w/v) dry milk followed by incubation for 2 hrs with the respective primary antibody diluted in TBS buffer. Blots were then washed three times for 10 min each in TBST and incubated with anti-rabbit (1:25000) or anti-mouse (1:10000) IgG horseradish ...
Hox genes encode transcriptional regulatory proteins that are largely responsible for establishin... more Hox genes encode transcriptional regulatory proteins that are largely responsible for establishing the body plan of all metazoan organisms. A subset of Hox genes is expressed during the period of organogenesis and into adulthood. hoxb-13 is a recently-described member of the Hox gene family that is expressed in the spinal cord, hindgut, and urogenital sinus during embryogenesis. Northern blot and in situ hybridization analyses of hoxb-13 expression in adult mouse tissues were performed. hoxb-13 mRNA is restricted to the prostate gland and distal colon in adult animals. In situ hybridization of mouse prostate tissue demonstrated that hoxb-13 is expressed in the epithelial cells of the ventral, dorsal, lateral, and anterior prostate lobes. Accumulation of hoxb-13 mRNA is not diminished following castration. These data demonstrate that hoxb-13 expression is androgen-independent in mouse prostate glands. The identification of hoxb-13 as an androgen-independent gene expressed in adult mouse prostate epithelial cells provides a new potential target for developing therapeutics to treat advanced prostate cancer.
Backgrounds and Objectives Small Heat Shock Proteins (HSPs) display chaperone-like activities and... more Backgrounds and Objectives Small Heat Shock Proteins (HSPs) display chaperone-like activities and protect cells against apoptosis and ischemia/reperfusion (I/R) injury. HSPB2 is expressed at high l...
... OF HSF [9] [9] P urificationof Heat Shock T ranscription F actorof Drosophila By MIN ZHONG, J... more ... OF HSF [9] [9] P urificationof Heat Shock T ranscription F actorof Drosophila By MIN ZHONG, JAN WISNIEWSKI, MICHAEL FRITSCH, GAKU MIZUGUCHI, ANDRAS OROSZ, PAUL ... 2 j. Clos, JT Westwood, PB Becker, S. Wilson, K. Lambert, and C. Wu, Cell63, 1085 (1990). ...
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