Sab4200181

Fibroblasts were lysed with ice-cold RIPA buffer (50 mM Tris-Cl, pH 8.0, 150 mM NaCl, 1% Nonidet P-40, 0.1% sodium dodecyl sulfate, 0.5% sodium deoxycholate, protease inhibitors, and phosphatase inhibitors). The samples were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE) and transferred to a PVDF membrane (Bio-Rad, Hercules, CA). Membranes were blocked with 5% non-fat milk and then incubated with anti-HSD17B4 (1:250, HPA021302; Atlas Antibodies, Stockholm, Sweden), anti-PMP70 (1:500, SAB4200181; Sigma-Aldrich, St. Louis, MO), anti-ß-actin (1:2000, sc-47778; Santa Cruz Biotechnology, Dallas, TX), and anti-α tubulin (1:25000, 11224-1-AP; Proteintech, Rosemont, IL) primary antibodies. The immunoreactive bands were detected with LAS-4000 (GE Healthcare, Chicago, IL). The quantitative densitometric analyses were performed with Image Quant LAS 4000 software (GE Health care). Blue native PAGE (BN-PAGE) analysis was performed using the NativePAGE Novex Bis-Tris Gel System (Life Technologies, Carlsbad, CA). We used the NativePAGE 4–16% Bis-Tris Protein Gel (Life Technologies). Fibroblasts were lysed with ice-cold lysis buffer (50 mM bis-tris [HCl], pH 7.2, 50 mM NaCl, 10% glycerol, 0.001% Ponceau S, 1% digitonin, protease inhibitors, and phosphatase inhibitors).

Fibroblasts were fixed with 4% paraformaldehyde and stained with anti-HSD17B4 (1:50, HPA021302; Atlas Antibodies) and anti-PMP70 (1:250, SAB4200181; Sigma-Aldrich) antibodies. Anti-rabbit IgG Alexa Fluor 594 and anti-mouse IgG Alexa Fluor 488 (1:500; Thermo Fisher Scientific) were used as secondary antibodies. Immunostained cells were examined using a confocal microscope (LSM800; Carl Zeiss, Jena, Germany). Intensities were analyzed using ImageJ.

Cells were prepared for indirect IF as previously described,²⁹ (link) mounted onto slides using ProLong Gold Antifade Reagent with DAPI (Invitrogen, Waltham, MA, USA), and visualized using a Leica DMI600 microscope with a DFC345FX camera and LASX software (Leica, Richmond Hill, ON, Canada). Primary antibodies used were 1:300 rabbit anti-PTS1 (generated and gifted by Dr. Steven Gould, Johns Hopkins University, Baltimore, MD, USA), 1:150 mouse anti-human ABCD3 (SAB4200181; Sigma-Aldrich, St. Louis, MO, USA), and 1:300 rabbit anti-HsPEX5 (generated and gifted by Dr. Gabriele Dodt, University of Tübingen, Germany). Secondary antibodies used were 1:400 Alexa Fluor 488 anti-rabbit (A21206; Invitrogen, Waltham, MA, USA) and 1:300 Alexa Fluor 594 anti-mouse (A11005; Invitrogen, Waltham, MA, USA).