Ripa buffer

Cells were incorporated with haemagglutinin (HA)‐ubiquitin and the previously described plasmids, then treated with MG132 (Yeasen) for 6 h to inhibit ubiquitin‐proteasome‐dependent dysregulation of protein. Cell lysates were made in RIPA buffer (Yeasen), prior to overnight incubation in anti‐Flag antibody or IgG at 4°C. The resulting immunoprecipitates were assayed using an anti‐HA‐Tag antibody‐mediated Western blotting. The details of antibodies used can be found in Supplementary Table S2.

Cycloheximide (CHX), polybrene, polyethylenimine (PEI), puromycin, and Oil Red O were from Sigma–Aldrich (St. Louis, MO, U.S.A.), MG132 was from BD Biosciences (Franklin Lakes, NJ, U.S.A.), and Polyjet, Lipo2000 and TRIzol reagent were from Invitrogen (Carlsbad, CA, U.S.A.). RIPA buffer and PCR 2X mix were from Yeasen (Shanghai, China). Rapamycin was from MCE (NJ, USA). AICAR was from CSNpharm (Chicago, MI, USA). Sodium caprylate was from Sangon (Shanghai, China). Antibodies against Flag tag and α-tubulin were from Sigma–Aldrich; antibodies against Myc tag and mouse IgG were from Santa Cruz Biotechnology (Santa Cruz, CA, U.S.A.); antibodies against GFP, HUWE1, HSP90, CREB, phosphorylated CREB, and TP53 were from Cell Signaling Technology (Boston, MA, USA); antibodies against HA and GAPDH were from ABclonal (Boston, MA, U.S.A.); antibodies against PAQR9, PPARα, Alexa Fluor 546 goat anti-mouse IgG, and Alexa Fluor 546 goat anti-rabbit IgG were from Abcam (Cambridge, U.K.); antibody against ubiquitin was from Santa Cruz Biotechnology (TX,U.S.A.).

Cells were treated with 0, 5, 10 or 20 µM PQR309 for 72 h and then lysed in RIPA buffer (Shanghai Yeasen Biotechnology, Co., Ltd.) for ~20 min on ice. BCA was used to test the concentrations of each sample then, the protein samples were loaded onto 10 or 12% SDS-PAGE for 40 µg per lane and electro-transferred to PVDF membranes (Merck KGaA) for 60 or 90 min. After the transfer, the membrane was blocked with 5% skim milk and then incubated with the primary antibody (all used at 1:1,000) overnight at 4°C. Subsequently, the membranes were incubated with Alexa Fluor 680/790-labelled goat anti-rabbit or goat anti-mouse IgG secondary antibodies (cat. nos. 926-68021 and 926-68020; Li-COR Biosciences) (all used at 1:1,000; LI-COR Biosciences) for 1 h. The bands were visualized using the LI-COR Odyssey Infrared Imaging System (Li-COR Biosciences) and the results were normalized to GAPDH.

Erythrocytes were lysed in RIPA buffer (Yeasen, Shanghai, China) containing a protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA). The supernatants of lysates were collected by centrifugation (12,000 rpm, 4 °C, 15 min). Activities of antioxidant enzymes, including GPX, SOD, CAT, and GR, as well as the contents of GSH and MDA were detected by specific assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) and normalized to Hb concentrations.

Cells were lysed in radio immunoprecipitation assay (RIPA) buffer (Yeasen, Shanghai, China), and protein was collected by centrifugation (4°C, 12,000 × g, 10 min). Protein concentrations were determined by bicinchoninic acid (BCA) protein assay kit (Yeasen, Shanghai, China). Proteins were separated by 15% SDS-PAGE and subsequently transferred onto polyvinylidene fluoride (PVDF) membranes. Membranes were blocked with 5% non-fat milk in Tris-buffered saline Tween 20 (TBST) for 2 h with gentle shaking at room temperature, then incubated overnight with the following primary antibodies: anti-LC3B (1:1,000; Abcam, Cambridge, MA, USA), anti-SQSTM1 (1:1,000; Abcam, Cambridge, MA, USA), anti-PDCD4 (1:1,000; Abcam, Cambridge, MA, USA), and anti-β-tubulin (1:6,000; Abcam, Cambridge, MA, USA). The membranes were washed three times with TBST for 10 min and incubated for 1 h with secondary antibodies (1:2,000, Proteintech, Rosemont, IL, USA) at room temperature. Protein bands were scanned using a Tanon 4600SF (Tanon, Shanghai, China). The density of protein bands was quantified with Image-Pro Plus 6.0 software, and the ratio of target protein to β-tubulin, which reflects the changes in expression levels, was calculated.