Protocol detail

Constructing and Characterizing Hsd17b4 Domains

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All plasmids in this study were constructed through polymerase chain reaction (PCR)-base strategy and sequenced to confirm their identity. Full cDNA sequence of mouse Hsd17b4 was purchased from Darmacon (USA) (MMM4769-202765308). Full sequence Hsd17b4 was constructed in the pEBB-Flag and pEBB-GFP vector (Min et al., 2020 (link)). The untagged protein expressing construct was generated as well. In order to identify PS binding region of Hsd17b4, the three domains of Hsd17b4 were constructed in the pEBG-GST vector. GST-Hsd17b4^1-305, GST-Hsd17b4^321-621, and GST-Hsd17b4^633-730 contain the hydroxyacyl-CoA dehydrogenase domain, Enoyl-CoA hydratase 2 domain, and the SCP2-like domain of Hsd17b4. HA-Tim-4^IgV was previously reported (Lee et al., 2019 (link)).
The antibodies used in this study were anti-Hsd17b4 (15116-1-AP [Proteintech, USA] and NBP2-46005 [Novus, USA]), anti-GST (SC-138; Santa Cruz Biotechnology, USA), anti-Catalase (ab209211; Abcam, UK), anti-Actin (SC-47778; Santa Cruz Biotechnology), anti-Pex5 (GTX109798; GeneTex, USA), anti-Pmp70 (ab3421; Abcam), anti-HA (sc-7392; Santa Cruz Biotechnology), Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488, and Goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 555 (A11008 and A21422; Thermo Fisher Scientific, USA).

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Lee S.A., Lee J., Kim K., Moon H., Min C., Moon B., Kim D., Yang S., Park H., Lee G., Park R, & Park D. (2021). The Peroxisomal Localization of Hsd17b4 Is Regulated by Its Interaction with Phosphatidylserine. Molecules and Cells, 44(4), 214-222.

Publication 2021

SC-138 anti-Catalase ab209211 anti-Actin anti-Pex5 ab3421 sc-7392 A21422

Actin Alexa fluor 488 Alexa fluor 555 Anti igg Antibodies Antibody Catalase Cdna Dehydrogenase Enoyl coa hydratase 2 Goat Hsd17b4 Mouse Novus Plasmids Polymerase chain reaction Protein Rabbit Vector

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Variable analysis

independent variables

Full cDNA sequence of mouse Hsd17b4
Three domains of Hsd17b4 (hydroxyacyl-CoA dehydrogenase domain, Enoyl-CoA hydratase 2 domain, and the SCP2-like domain)
HA-Tim-4^IgV

dependent variables

Hsd17b4 binding to phosphatidylserine (PS)

control variables

Plasmids constructed through polymerase chain reaction (PCR)-based strategy
Plasmids sequenced to confirm their identity
Antibodies used for detection and identification

positive controls

HA-Tim-4^IgV (previously reported)

negative controls

Not explicitly mentioned

Annotations

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