Publication protocol
HeLa cells, MEFs or HEK293E were seeded in 6- or 12-well plates 24 (HeLa) or 48 (MEFs) hours prior treatments. After treatments, cells were washed in ice-cold 1× PBS then lysed in RIPA buffer (150 mM NaCl, 1% NP40, 0.5% NaDoC, 0.1% SDS, 50 mM Tris pH 7.4, supplemented with 1× Halt Protease & Phosphatase inhibitor cocktail (Thermo Scientific) in ddH2O) plus 50 mM N-ethylmaleimide, which interacts with reduced cysteines and prevents new disulphide bond formation during lysis procedures and simultaneously inactivates deubiquitylating enzymes. Cell lysates were then centrifuged at 4 °C at 13,000 rpm for 10 min to remove insoluble cellular components. Protein concentration was measured by Bradford assay using the DC Protein Assay (BioRad) and a FLUOstar Omega plate reader (BMG Labtech). Samples were prepared by boiling in SDS-Loading buffer (BioRad) at 100 °C for 5 min in the presence or absence of 2.5% β-ME (β-mercaptoethanol, Sigma). An amount of 20–40 μg of proteins was run on 10–12% Tris-Glycine SDS-PAGE gels and transferred to Immobilon-P (Millipore) PVDF membranes using a Trans-Blot SD Semi-Dry Transfer Cell (BioRad). Blots were incubated with a blocking solution (PBS containing 5% fat-free dry milk, 0.1% Tween-20) for 1 h. After washing with PBS, blots were incubated with primary antibodies diluted in blocking solution at 4˚C overnight. Blots were then washed three times, 5 min each: one time with PBS, once in PBS with 0.1% Tween-20 and one more time in PBS. Then, blots were incubated with secondary HRP-conjugated antibodies α-mouse or α-rabbit (Sigma, #A2554 and #A0545, 1:5000) or α-guinea pig (Dako, #P0141, 1:1000) in blocking solution for 1 h at room temperature. Blots were then washed three times as above. Blots were incubated for 5 min with the Clarity Western ECL Substrate (BioRad) and signals were detected by chemiluminescence using a LAS-4000 CCD camera system (Fujifilm). The following primary antibodies were used: guinea pig α-p62 (Progen Biotechnik #GP62-C, 1:2000), mouse α-GFP (Santa Cruz Biotechnology #sc-9996, 1:1000), mouse α-Ubiquitin (LifeSensors #VU101, 1:1000), rabbit α-PRDX349 (1:1000), α-PRDX-SO3 (Abcam #ab16830, 1:2000), mouse α-LC3 (Enzo Life Sciences #ALX-803-081-C100, 1:1000), mouse α-FLAG (Sigma #F1804, 1:2000), rabbit α-FLAG (Sigma #F7425, 1:2000), rabbit α-GAPDH (CST #5174, 1:10,000), rabbit α-actin (CST #4970, 1:2000), rabbit α-Drosophila tubulin (Abcam #ab179513, 1:2000), Ref(2)P (Abcam #ab178440, 1:1000) or (50, a kind gift from Ioannis Nezis, Warwick University, 1:500), α-Drosophila Atg851 (a kind gift from Ivana Bjedov, UCL, 1:1000), rabbit α-Nrf2 (CST #12721, 1:500) and rabbit α-Histone H3 antibody (CST #9715, 1:2000). Uncropped versions of western blots are presented in Supplementary Fig. 14.
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