Fig 1: (A) Testosterone clearance in AKR1D1-002 (blue bar), AKR1D1-001 (red bar), AKR1D1-006 (green bar) or empty vector- (EV, black bar) transfected HEK293 cells after 24 h of testosterone treatment (200 nM). (B) Testosterone-induced androgen receptor (AR) activation in AKR1D1-002 (blue bar), AKR1D1-001 (red bar), AKR1D1-006 (green bar) overexpressing and empty vector (EV, black bar) transfected HEK293 cells after 24 h of testosterone treatment (200 nM). (C) Androstenedione clearance in AKR1D1-002 (blue bar), AKR1D1-001 (red bar), AKR1D1-006 (green bar) or empty vector- (EV, black bar) transfected HEK293 cells after 24 h of androstenedione treatment (200 nM). (D) Representative UHPLC-MS/MS chromatogram of 5α- and 5β-androstanedione in empty vector (EV, black line), AKR1D1-002 (blue line), AKR1D1-001 (red line) and AKR1D1-006- (green line) transfected HEK293 cells after 24 h of androstenedione treatment (200 nM). (E) Media 5α- and 5β-androstanedione levels in empty vector (EV, black line), AKR1D1-002 (blue line), AKR1D1-001 (red line) and AKR1D1-006- (green line) transfected HEK293 cells after 24 h of androstenedione treatment (200 nM). (F) Androstenedione-induced androgen receptor (AR) activation in AKR1D1-002 (blue bar), AKR1D1-001 (red bar), AKR1D1-006 (green bar) overexpressing and empty vector (EV, black bar) transfected HEK293 cells after 24 h of androstenedione treatment (200 nM). Data are presented as mean ± s.e. of n = 5 experiments, performed in duplicate, *P < 0.05, **P < 0.01, ***P < 0.001 compared to empty vector (EV) transfected controls.
Fig 2: (A) Schematic representation demonstrating the mechanism of TaqMan qPCR targeting the different AKR1D1 splice variants using specific exon–exon junction primers. (B) Relative mRNA expression levels of AKR1D1-001, AKR1D1-002 and AKR1D1-006 splice variants in male and female human liver biopsies (n = 8), Huh7 (n = 4) and HepG2 (n = 4) hepatoma cell lines. mRNA overexpression levels of (C) AKR1D1-002, (D) AKR1D1-001, and (E) AKR1D1-006 in HEK293 cells, confirmed using multiple exon–exon junction Taqman primer assays (n = 5). mRNA expression data are presented as mean ± s.e., performed in duplicate.
Fig 3: (A) Clustal Omega multiple alignment of the amino acid sequences of the human AKR1D1 splice variants -002, -001 and -006. (B) AKR1D1-001.NADP+.Cortisone Ternary Complex (PDB 3CMF). Magneta shows deletion of residues 153-193; NADP+ (red) and cortisone (blue). There are two monomers per asymmetric unit. (C) AKR1D1-006.NADP+.Cortisone Ternary Complex (PDB 3CMF). Magneta shows deletion of exon 8 (residues 285–326); NADP+ (red) and cortisone (blue). There are two monomers per asymmetric unit. Colour coding in figure A represents amino acid physicochemical properties: RED: Small (small + hydrophobic (aromatic)); BLUE: Acidic; MAGENTA: Basic – H; GREEN: Hydroxyl + sulfhydryl + amine + G; ✶ positions which have a single, fully conserved residue. B and C made in PyMol.
Fig 4: (A) Protein expression levels of AKR1D1-002, AKR1D1-001 and AKR1D1-006, following overexpression in HEK293 cells, as measured by Western blotting. (B) Protein expression levels of AKR1D1-002, AKR1D1-001 and AKR1D1-006 following overexpression in HEK293 cells for 48 h, and subsequent treatment with either DMSO (vehicle, black bars) or 20 µM MG-132 (proteasomal inhibitor) for 3 h (maroon bars) and 6 h (teal bars). Representative Western blotting images are shown from one biological replicate, however, formal quantification was performed in n = 4 replicates. Data are presented as mean ± s.e. *P < 0.05, **P < 0.01, compared to empty vector (EV) transfected controls.
Fig 5: (A) AKR1D1 transcript levels in human adipose, adrenal gland, breast, brain, liver, skeletal muscle, ovary and testicular tissues.$ (B) Transcript expression levels of AKR1D1 splice variants in human liver.$ (C) Transcript expression levels of AKR1D1 splice variants in human testes.$ $Data extracted from https://www.gtexportal.org (GTEx). GTEx-extracted expression values are shown as transcripts per million (TPM). Box plots are shown as median and 25th and 75th percentiles. Points displayed are outliers 1.5 times above or below the interquartile range.
Supplier Page from OriGene Technologies for AKR1D1 (NM_005989) Human Untagged Clone