Ab75060

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.

Western blotting assays were performed by the protocol described in our previous report [7 (link)]. The following antibodies are used for immune blotting assays: monoclonal anti-Flag (M185-3L, MBL), Akt (610860, BD Biosciences), β-Actin (sc-4778, Santa Cruz) and the cell signaling antibodies (p-Akt, ERK, and p-ERK). GNPAT antibody was purchased from Abcam (ab75060).