Nrf2 16396 1 ap

Cells were lysed with 10 μL/mL Radio-Immunoprecipitation assay Lysis Buffer containing protease inhibitors (Beyotime), centrifuged at 14,000 × g, and quantified by bicinchoninic acid protein assay (Beyotime). Total protein (20 μg) was loaded into a sodium lauryl sulfate polyacrylamide gel and separated. Then, the sample was electroblotted onto a polyvinylidene fluoride membrane (Millipore, Billerica, MA, USA), blocked with 5% skim milk, and incubated with the following primary antibodies: β-actin (A5441, MilliporeSigma), KLF6 (14716-1-AP, Proteintech), Nrf2 (16396-1-AP, Proteintech), NF-κB (14220-1-AP, Proteintech), p-NF-κB (8242, Cell Signaling Technology), HO-1 (10701-1-AP, Proteintech), Bax (50599-2-Ig, Proteintech), Bcl-2 (12789-1-AP, Proteintech), and Ki-67 (27309-1-AP, Proteintech). The membrane was then incubated with the secondary antibody conjugated with horseradish peroxidase (Beyotime), developed by enhanced chemiluminescence kit (Amersham Pharmacia Biotech, Little Chalfont, UK) and imaged by ChemiDoc XRS imaging system. Image J image analysis software was used for data analysis. Three independent replicates were performed for each group.

Cells were lysed using Nonidet-P40 lysis buffer (1% NP-40, 150 mM NaCl, 10% glycerol, 1 mM EDTA, and 20 mM Tris-HCl [pH 7.4]). Then, the membranes were blocked with 5% skim milk in TBS-Tween 20 (TBST), incubated with primary antibodies overnight at 4 °C and then incubated with secondary antibodies for 1 h at room temperature. For the detection of ELOVL5 protein, the samples were not boiled as previously described^{8 (link)}. Primary antibodies against ACC (3662, 1:1000), ACC-p (3661,1:1000), FASN (3180,1:1000), FTH1 (3998,1:1000), S6K (9202,1:1000) and S6K-p (9205,1:1000) were purchased from Cell Signaling Technology (USA). SREBP1 (557036,1:1000) was purchased from BD Biosciences (USA). ELOVL5 (sc-398653,1:2000), ACSL4 (sc-365230,1:1000), FSP1 (sc-377120,1:1000), PEBP1 (sc-376925,1:2000) and HSP90 (sc-13119,1:3000) were procured from Santa Cruz (USA). GPX4 (ab125066,1:2000) and Na/K ATPase (ab76020,1:1000) were purchased from Abcam (UK). NRF2 (16396-1-AP,1:1000) and FADS1 (10627-1-AP,1:1000) were obtained from Proteintech (USA). LPCAT3 (16-999,1:1000) was purchased from ProSci (USA). Flag (F3165,1:3000), β-actin (A5316,1:10,000) and α-tubulin (T9026,1:10,000) were purchased from Sigma-Aldrich (USA). Detailed information on materials and cells used in our study is presented in Supplementary Table 4.