Journal: The Journal of Biological Chemistry

Article Title: Dehydrogenase reductase 9 (SDR9C4) and related homologs recognize a broad spectrum of lipid mediator oxylipins as substrates

doi: 10.1016/j.jbc.2021.101527

Figure Lengend Snippet: Expression of SDR9C proteins in Sf9 microsomes. A , Coomassie Blue-stained SDS-PAGE gel. Fifty micrograms of Sf9 microsomes containing different recombinant proteins were loaded into each lane. RDH10 microsomes were loaded as a negative control. The positions of each recombinant protein are indicated by the red boxes . Based on scanning densitometry, the protein content of each SDR in microsomal preparations relative to human RDH16, which was set as 1, was as follows: 3.15-fold higher for human HSD17B6, 1.63-fold higher for rat RDH7, and 1.6-fold higher for human DHRS9. B , Western blot analysis of the same set of microsomal preparations (0.5 μg/lane) using human RDH16 (RoDH4) antibodies (N-terminal M17, 1:5000) . Note that the antibodies detect human HSD17B6 protein and also weakly react with rat RDH7. C , Western blot analysis of the same set of microsomal preparations (0.5 μg/lane) using DHRS9 antibodies (Abclonal, 1:2000).

Article Snippet: Blots were incubated overnight at 4 °C in blocking buffer with the following primary antibodies: DHRS9 rabbit polyclonal antibody (#A6324, Abclonal) at 1:2000 dilution, cytochrome P450 reductase antibodies (#AB1257, Chemicon International) at 1:4000 dilution or with custom-made rabbit polyclonal DHRS9 antibodies (1:250), and cytochrome p450 antibodies ab #13513 (Abcam, 1:1000).

Techniques: Expressing, Staining, SDS Page, Recombinant, Negative Control, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Dehydrogenase reductase 9 (SDR9C4) and related homologs recognize a broad spectrum of lipid mediator oxylipins as substrates

doi: 10.1016/j.jbc.2021.101527

Figure Lengend Snippet: Substrate specificity of human DHRS9 (SDR9C4)

Article Snippet: Blots were incubated overnight at 4 °C in blocking buffer with the following primary antibodies: DHRS9 rabbit polyclonal antibody (#A6324, Abclonal) at 1:2000 dilution, cytochrome P450 reductase antibodies (#AB1257, Chemicon International) at 1:4000 dilution or with custom-made rabbit polyclonal DHRS9 antibodies (1:250), and cytochrome p450 antibodies ab #13513 (Abcam, 1:1000).

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: Dehydrogenase reductase 9 (SDR9C4) and related homologs recognize a broad spectrum of lipid mediator oxylipins as substrates

doi: 10.1016/j.jbc.2021.101527

Figure Lengend Snippet: Characterization of mouse DHRS9 activities towards oxylipins. A , Western blot analysis of mouse DHRS9 expression in HEK 293 cells. HEK 293 cells were transiently transfected with mouse DHRS9 expression construct. HEK293 microsomes (30 μg) containing DHRS9 protein were analyzed by Western blotting using DHRS9 antibodies (Abclonal, 1:2000). Immunodetection with cytochrome P450 reductase antibodies (#13513 Abcam, 1:1000) was used for a gel loading control. B – H , activities of mouse DHRS9. Microsomes (5 μg) isolated from HEK293 cells transfected with either empty vector ( black traces ) or mouse Dhrs9 expression construct ( red traces ) were incubated with substrates (1 μM each) as indicated. The reaction products were extracted and analyzed as described under . Top panels —chromatograms extracted at the λ max of the substrate, bottom panels —at λ max of the product. Note the appearance of the oxidized products in the presence of DHRS9. Insets , spectra for substrates ( black ) and products ( red ).

Article Snippet: Blots were incubated overnight at 4 °C in blocking buffer with the following primary antibodies: DHRS9 rabbit polyclonal antibody (#A6324, Abclonal) at 1:2000 dilution, cytochrome P450 reductase antibodies (#AB1257, Chemicon International) at 1:4000 dilution or with custom-made rabbit polyclonal DHRS9 antibodies (1:250), and cytochrome p450 antibodies ab #13513 (Abcam, 1:1000).

Techniques: Western Blot, Expressing, Transfection, Construct, Immunodetection, Control, Isolation, Plasmid Preparation, Incubation

Journal: The Journal of Biological Chemistry

Article Title: Dehydrogenase reductase 9 (SDR9C4) and related homologs recognize a broad spectrum of lipid mediator oxylipins as substrates

doi: 10.1016/j.jbc.2021.101527

Figure Lengend Snippet: Activity of mouse DHRS9 toward oxylipins

Article Snippet: Blots were incubated overnight at 4 °C in blocking buffer with the following primary antibodies: DHRS9 rabbit polyclonal antibody (#A6324, Abclonal) at 1:2000 dilution, cytochrome P450 reductase antibodies (#AB1257, Chemicon International) at 1:4000 dilution or with custom-made rabbit polyclonal DHRS9 antibodies (1:250), and cytochrome p450 antibodies ab #13513 (Abcam, 1:1000).

Techniques: Activity Assay

Journal: The Journal of Biological Chemistry

Article Title: Dehydrogenase reductase 9 (SDR9C4) and related homologs recognize a broad spectrum of lipid mediator oxylipins as substrates

doi: 10.1016/j.jbc.2021.101527

Figure Lengend Snippet: Generation of Dhrs9 −/− mice. A , diagram of Dhrs9 tm1(KOMP)Vlcg knockout allele. Velocigene KOMP Definitive Null Allele Design #13240 replaces 8577 bp of mouse chromosome 2 with Velocigene ZEN-UB1 cassette encoding promoterless LacZ reporter and human ubiquitin C promoter-driver neomycin selection marker. The generated allele lacks the complete protein-coding sequence of Dhrs9 gene and expresses LacZ under the control of endogenous Dhrs9 promoter. Coding exons (E2–E5) are shown as black rectangles , noncoding E1 and portions of E2 and E5—as gray rectangles . Positions of genotyping primers are indicated by the black arrows . B , validation of Dhrs9 gene knockout by Western blot analysis. Microsomes (80 μg) isolated from skin of two wild-type (WT) and two Dhrs9 KO mice were separated by SDS-PAGE and transferred to PVDF Immobilon–P membrane. The membrane was incubated with rabbit polyclonal DHRS9 antibodies (1:250 dilution) obtained from Dr Helen Everts at Texas Woman's University. Microsomes (20 μg) isolated from HEK 293 cells (HEK) transfected with mouse DHRS9 (mDHRS9) expression construct were used as a standard (St. DHRS9). Immunodetection with cytochrome p450 ab 13513 (Abcam, 1:1000) was used for a gel loading control.

Article Snippet: Blots were incubated overnight at 4 °C in blocking buffer with the following primary antibodies: DHRS9 rabbit polyclonal antibody (#A6324, Abclonal) at 1:2000 dilution, cytochrome P450 reductase antibodies (#AB1257, Chemicon International) at 1:4000 dilution or with custom-made rabbit polyclonal DHRS9 antibodies (1:250), and cytochrome p450 antibodies ab #13513 (Abcam, 1:1000).

Techniques: Knock-Out, Ubiquitin Proteomics, Selection, Marker, Generated, Sequencing, Control, Biomarker Discovery, Gene Knockout, Western Blot, Isolation, SDS Page, Membrane, Incubation, Transfection, Expressing, Construct, Immunodetection

Journal: The Journal of Biological Chemistry

Article Title: Dehydrogenase reductase 9 (SDR9C4) and related homologs recognize a broad spectrum of lipid mediator oxylipins as substrates

doi: 10.1016/j.jbc.2021.101527

Figure Lengend Snippet: Analysis of LTB4 and RvD1 dehydrogenase activity of microsomes isolated from wild-type (WT) versus Dhrs9 gene knockout (KO) mice. Microsomes (50 μg) of female mice (28–35 days old) isolated from the skin, lungs, and several tracheas pooled into three samples were incubated with 1 μM LTB 4 ( A ) or 1 μM RvD1 ( B ) and 1 mM NAD + for 1 h at 37 °C. The reaction products were analyzed by reversed-phase HPLC. The results are presented as mean ± S.D., ∗ p ≤ 0.05; ∗∗ p ≤ 0.01.

Article Snippet: Blots were incubated overnight at 4 °C in blocking buffer with the following primary antibodies: DHRS9 rabbit polyclonal antibody (#A6324, Abclonal) at 1:2000 dilution, cytochrome P450 reductase antibodies (#AB1257, Chemicon International) at 1:4000 dilution or with custom-made rabbit polyclonal DHRS9 antibodies (1:250), and cytochrome p450 antibodies ab #13513 (Abcam, 1:1000).

Techniques: Activity Assay, Isolation, Gene Knockout, Incubation