Journal: The Biochemical journal

Article Title: Characterization of subunit interactions in the hetero-oligomeric retinoid oxidoreductase complex

doi: 10.1042/BCJ20210589

Figure Lengend Snippet: A. Microsomal membranes were extracted with sodium carbonate buffer, pH 11.5 (Alk Extr), phosphate-buffered saline, pH 7.4 (PBS), or 1% Triton X-100 in PBS (1% Tri). Solubilized proteins (S) were separated from integral membrane proteins (P) by centrifugation. Distribution of proteins between the soluble and the membrane-bound fractions was analyzed by Western blotting as described under “Materials and Methods.” B. HEK 293 cells were transfected with wild type (WT) or mutant constructs using Lipofectamine 2000 (Invitrogen) transfection reagent according to the manufacturer’s protocol and harvested 24 h after transfection. After separation in SDS-PAGE and transfer to PVDF membranes, DHRS3 protein was detected using DHRS3 antibodies at a 1:2,000 dilution. RDH10 protein was detected using RDH10 antibodies at a 1:3,000 dilution.

Article Snippet: Western blots and antibodies Proteins from HEK 293 cell extracts or Sf9 microsomes were separated by SDS-PAGE and transferred to Amersham Hybond P PVDF membranes (Cytiva, Marlborough, MA).

Techniques: Saline, Membrane, Centrifugation, Western Blot, Transfection, Mutagenesis, Construct, SDS Page