Fig 1: Nrf2 and MafG occupy and transactivate the MARE-1 element at proximal promoter region of AHSP.(A) JASPAR analysis predicted a putative MafG/K binding site, MARE-1, located +304 to +315 bp downstream of the AHSP transcriptional initiation site. Sequences of the original MARE-1 and the mutant used in (F) are shown.(B) ChIP-seq data showing MafG and MafK binding status in the proximal promoter region of AHSP in K562 cells (data obtained from MafG: GSE92076 and MafK: GSE31477).(C and D) EMSA of the MARE-1. A probe containing the MARE-1 was incubated with purified MafG protein (C) or purified Nrf2 and MafG proteins (D). Excess unlabeled cold probe (200-fold) was included as indicated.(E) ChIP analysis of Nrf2, MafG and MafK occupancy at the MARE-1 site on the AHSP gene in a-globin-overloaded and control K562 cells (the data are presented as the mean ± SD; ns, Not significant; **, P < 0.01; n = 3 replicates).(F) Dual-luciferase reporter assays to determine the enhancer activity of the MARE-1-WT fragment (+52 to +552 bp from the AHSP transcriptional initiation site), the effect of the MARE-1 element mutation, and the ability of Nrf2 to transactivate the enhancer activity of the MARE-1-WT fragment. The pGL3-promoter vector with the SV40 promoter was used as a control (the data are presented as the mean ± SD; **, P < 0.01; ***, P < 0.005; n = 3 replicates).(G) Dual-luciferase reporter assays to determine the ability of MafK or MafG to cooperate with Nrf2 in transactivating the enhancer activity of the MARE-1-WT fragment (the data are presented as the mean ± SD; ns, ***, P < 0.005; n = 3 replicates).
Fig 2: AHSP is upregulated in ß-thalassemia models, and partially represses a-globin aggregation and ROS production.(A) Real-time PCR analysis of AHSP expression in a-globin-overloaded and control K562 cells. (the data are presented as the mean ± SD; **, P < 0.01; n = 3 replicates).(B) Representative Western blot analysis of Nrf2 and AHSP expression before and after Nrf2 knockdown in a-globin-overloaded and control K562 cells. AHSP and Nrf2 levels were assessed by densitometric quantification and normalized to ß-actin levels (the data are presented as the mean ± SD; *, P < 0.005; **, P < 0.01; ***, P < 0.005; n = 4 replicates).(C) Real-time PCR analysis of AHSP, GCLM and NQO1 expression following Nrf2 knockdown in a-globin-overloaded K562 cells (the data are presented as the mean ± SD; *, P < 0.005; **, P < 0.01; ***, P < 0.005; n = 3 replicates).(D) Representative Western blot analysis of AHSP expression in splenic Ter119+ cells isolated from ßIVS-2-654 thalassemic and wild-type mice. AHSP levels were assessed by densitometric quantification and normalized to ß-actin levels (the data are presented as the mean ± SD; ***, P < 0.005; WT, n = 3 mice; ßIVS-2-654, n = 3 mice).(E) Real-time PCR analysis of AHSP expression in splenic Ter119+ cells isolated from ßIVS-2-654 thalassemic and wild-type mice. (the data are presented as the mean ± SD; ***, P < 0.005; WT, n = 3 mice; ßIVS-2-654, n = 3 mice).(F) Real-time PCR analysis of AHSP and GCLM expression in bone marrow basophilic erythroblasts (Baso), polychromatic erythroblasts (Poly) and orthochromatic erythroblasts (Ortho) from wild-type and ßIVS-2-654 thalassemic mice. (the data are presented as the mean ± SD; **, P < 0.01; ***, P < 0.005; n = 3 replicates).(G-I) ßIVS-2-654 thalassemic mouse fetal liver (E14.5) derived erythroid cells were treated with tBHQ or DMSO during in vitro differentiation. (G) Representative Western blot analysis of insoluble and soluble a-/ß-globin, Nrf2 and AHSP protein levels. (H) Ratio of insoluble to soluble a-globin (the data are presented as the mean ± SD; ***, P < 0.005; n = 3 replicates). (I) ROS detection assay (the data are presented as the mean ± SD; *, P < 0.05; n = 3 replicates).(J-O) RNA interference of Nrf2 (shNrf2) or AHSP (shAHSP) in ßIVS-2-654 thalassemic mouse fetal liver (E14.5) derived erythroid cells. Control cells were transfected with shRNA targeting luciferase (shLuc). (J, M) Representative Western blot analysis of insoluble and soluble a-/ß-globin, Nrf2 (for Panel J only) and AHSP protein levels. (K, N) Ratio of insoluble to soluble a-globin levels (the data are presented as the mean ± SD; ***, P < 0.005; n = 3 replicates). (L, O) ROS detection assay (the data are presented as the mean ± SD; ***, P < 0.005; n = 3 replicates).
Fig 3: MafG facilitates Nrf2-mediated regulation of AHSP expression.(A) Representative Western blot analysis of Nrf2, MafG and AHSP expression in Nrf2-overexpressing and control K562 cells. MafG, AHSP and Nrf2 levels were assessed by densitometric quantification and normalized to ß-actin levels (the data are presented as the mean ± SD; ***, P < 0.005; n = 4 replicates).(B) Representative Western blotting analysis of Nrf2, MafG and AHSP expression in MafG-overexpressing and control K562 cells. MafG, AHSP and Nrf2 levels were assessed by densitometric quantification and normalized to ß-actin levels (the data are presented as the mean ± SD; **, P < 0.01; ***, P < 0.005; n = 5 replicates).(C) Representative Western blotting analysis of Nrf2 and MafG expression in K562 cells with or without tBHQ treatment for 6 h. Nrf2 and MafG levels were assessed by densitometric quantification and normalized to ß-actin levels (the data are presented as the mean ± SD; ***, P < 0.005; n = 4 replicates).(D-E) Representative Western blotting analysis of Nrf2, MafG and AHSP expression (D) and Real-time PCR analysis of AHSP, GCLM and NQO1 expression (E) in MafG-overexpressing K562 cells with or without tBHQ treatment for 6 h (the data are presented as the mean ± SD; *, P < 0.05; **, P < 0.01; ***, P < 0.005; D: n = 3 replicates; E: n = 5 replicates).(F) Representative Western blot analysis of Nrf2, MafG and AHSP expression in Nrf2 and MafG double-overexpressing and control K562 cells. Nrf2, MafG and AHSP levels were assessed by densitometric quantification and normalized to ß-actin levels (the data are presented as the mean ± SD; **, P < 0.01; ***, P < 0.005; n = 3 replicates).
Fig 4: The Nrf2 agonist tBHQ stimulates AHSP expression in wild-type mouse Ter119+ cells.(A) FACS analysis of the magnetic cell sorting efficiency and purity of Ter119+ cells isolated from mouse spleens.(B) Real-time PCR analysis of AHSP and GCLM expression levels in splenic Ter119+ cells from wild-type mice with or without in vitro tBHQ treatment for 6 h (the data are presented as the mean ± SD; **, P < 0.01; n = 3 replicates).(C) Representative Western blot analysis of AHSP expression in splenic Ter119+ cells from wild-type mice with or without in vitro tBHQ treatment for 6 h. AHSP levels were assessed by densitometric quantification and normalized to ß-actin levels (the data are presented as the mean ± SD; **, P < 0.01; n = 3 replicates).(D) Representative Western blot analysis of AHSP expression in splenic Ter119+ cells from wild-type mice with or without tBHQ treatment via tail vein injection. AHSP levels were assessed by densitometric quantification and normalized to ß-actin levels (the data are presented as the mean ± SD; **, P < 0.01; n = 3 replicates).(E-F) Immunofluorescence analysis of Nrf2 nuclear translocation (E) and real-time PCR analysis of AHSP and GCLM expression (F) in bone marrow Ter119+ cells from wild-type mice with or without tBHQ treatment via intraperitoneal injection (the data are presented as the mean ± SD; ***, P < 0.005; n = 3 replicates).
Fig 5: Nrf2 enhances AHSP expression in K562 cells.(A) Real-time PCR analysis of the expression of AHSP, NQO1 and GCLM in Nrf2-overexpressing and control K562 cells (the data are presented as the mean ± SD; *, P < 0.05; **, P < 0.01; ***, P < 0.005; n = 3 replicates).(B) Representative Western blot analysis of AHSP expression in Nrf2-overexpressing and control K562 cells. AHSP and Nrf2 levels were assessed by densitometric quantification and normalized to ß-actin levels (the data are presented as the mean ± SD; ***, P < 0.005; n = 4 replicates).(C) Immunofluorescence analysis of Nrf2 nuclear translocation in tBHQ-treated K562 cells.(D) Representative Western blot analysis of AHSP and Nrf2 expression in tBHQ-treated and control K562 cells. AHSP and Nrf2 levels were assessed by densitometric quantification and normalized to ß-actin levels (the data are presented as the mean ± SD; **, P < 0.01; ***, P < 0.005; n = 5 replicates).(E) Real-time PCR analysis of AHSP, NQO1 and GCLM expression in tBHQ-treated and control K562 cells (the data are presented as the mean ± SD; **, P < 0.01; ***, P < 0.005; n = 3 replicates).(F) Representative Western blot analysis of Nrf2 and AHSP expression in tBHQ-treated control and Nrf2-knockdown K562 cells. AHSP and Nrf2 levels were assessed by densitometric quantification and normalized to ß-actin levels (the data are presented as the mean ± SD; **, P < 0.01; n = 4 replicates).(G) Real-time PCR analyses of AHSP expression in tBHQ-treated K562-shNrf2 and shGFP control cells (the data are presented as the mean ± SD; ***, P < 0.005; n = 3 replicates).
Supplier Page from OriGene Technologies for AHSP Rabbit Polyclonal Antibody