Protein assay

The cells were lysed using RIPA buffer (radio-immunoprecipitation assay lysis buffer, BOSTER, China) supplemented with protein phosphatase inhibitors after
transfection for 48 h. Cell lysate was centrifuged at $\mathrm{12}\mathrm{000}\times g$ for 5 min at
4  ${}^{\circ }$ C and the supernatants were collected. The concentration of protein was
determined with a modified BCA (bicinchoninic acid protein quantitation assay) protein assay
(KeyGEN BioTECH, China) using a microplate spectrophotometer (BioTek Instruments EON,
USA). Equal concentrations of various proteins were resolved by SDS-PAGE and then
transferred onto PVDF (polyvinylidene fluoride) membranes (Bio-Red
Laboratories, Inc., USA). After blocked with 1 % BSA (bovine serum albumin) for 2 h, the PVDF membrane was washed with TBST and incubated with rabbit
polyclonal anti-ACSL5 antibody (Abcam, USA) and $\mathit{\beta }$ -actin (Abcam, USA) over
night at 4  ${}^{\circ }$ C. After being washed with TBST, the membrane was incubated with
enhanced chemiluminescent HRP-conjugated (horseradish peroxidase-conjugated)
anti-rabbit secondary antibody (BioWorld, USA) for 1.5 h at room temperature. The
membrane was washed with TBST and the developer (Invitrogen, USA) was uniformly dropped
on the membrane. The membrane was exposed using a chemiluminescence imager (Tanon,
Shanghai, China) after 3 min of reaction. The gray values of protein bands were digitized
by Image-Pro Plus.

Five days after transfection, proteins were isolated. The protein concentrations were determined by protein assay (KeyGEN, China). Electrophoresis on a 12% SDS polyacrylamide gel was performed and the protein was transferred to a PVDF membrane (Millipore, Germany). Protein bands were detected using an enhanced chemiluminescence system (Pierce Biotechnology, USA) with antibodies (Santa Cruz, USA).