Pa5 60311

Protein extracts were prepared using lysis buffer (8 mol/L urea, 75 mmol/L NaCl, 50 mmol/L Tris, pH 8.2, 1% [vol/vol] EDTA-free protease inhibitor, 1 mmol/L NaF, 1 mmol/L β glycerophosphate, 1 mmol/L sodium orthovanadate, 10 mmol/L sodium pyrophosphate, 1 mmol/L PMSF) containing a 1% (wt/vol) protease inhibitor mixture (ThermoScientific, Waltham, MA, USA). The proteins were separated by SDS-PAGE. The membranes were blocked and incubated with indicated primary antibodies. The following primary antibodies were used: anti-PEX16 (1∶1000; PA5-60311, Invitrogen), anti-MRPS22 (1∶1000; 10984-1-AP, Proteintech), anti-PEX14 (1∶1000; 10594-1-AP, Proteintech), anti-GNPAT (1∶1000; ab75060, Abcam), anti-AGPS (1∶1000; ab184186, Abcam), anti-HSD17B4 (1∶1000; 15116-1-AP, Proteintech), anti-β-actin (1∶5000; AC026, ABclonal, Wuhan, Hubei, China). The specific antibody for PEX3 was generated according to the published method^{[24 (link)]}. Following secondary antibody incubation, detected protein signals were visualized with SuperSignal West Femto Chemiluminescent Substrate (Thermo Scientific) on the Tanon 5200 image system. Intensities were analyzed with Image J and GraphPad Prism.

Testes were directly embedded in tissue freezing medium, sectioned at 5 µm, and fixed with 4% paraformaldehyde. Slides were blocked with bovine serum (Sigma), and stained with primary antibodies. The primary antibodies used were as follows: anti-PEX16 (1∶200; PA5-60311, Invitrogen), anti-ABCD1 (1∶200; 11159-1-AP, Proteintech, Manchester, UK), anti-ABCD3 (1∶200; PA1-650, Invitrogen), anti-TEX14 (1∶500; ab41733, Abcam, Cambridge, UK), anti-DDX4 (1∶200; ab27591, Abcam), anti-γ-H2AX (1∶200; ab26350, Abcam), anti-SOX9 (1∶200; AB5535, Millipore, Billerica, MA, USA), anti-PLZF (1∶200; AF2944, R&D Systems, Minneapolis, MN, USA). Secondary antibodies were FITC- or TRITC-conjugated goat anti-mouse or anti-rabbit IgG and donkey anti-goat IgG (1∶1000; Beijing Zhongshan Biotechnology Co., Beijing, China). The slides were visualized using a ZEISS LSM800 (ZEISS, Jena, Germany) microscope.