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6 protocols using histone 3

1

Protein Extraction and Western Blot Analysis

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Total proteins, cytoplasmic proteins, and nuclear proteins were extracted from cultured cells using the corresponding kits (Beyotime, Shanghai, China) according to the manufacturer's instructions. The protein samples were incubated with the following primary antibodies: TFRC (1 : 500), FTL (1 : 500), DMT1 (1 : 1000), MTF1 (1 : 500), JNK (1 : 1000), beta-actin (1 : 5000), GAPDH (1 : 1000), and Histone 3 (1 : 1000) (all obtained from Proteintech), FPN (1 : 1000; Novus Biologicals), and Phospho-JNK (1 : 1000; Cell Signaling Technology). This was followed by incubation with a horseradish peroxidase- (HRP-) conjugated secondary antibody (1 : 2000; Abcam). beta-actin, GAPDH, and Histone 3 were used for normalization.
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2

Investigating Cell Signaling Pathways

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Antibodies for Akt, Akt-Ser(P)-473, Akt-Thr(P)-308, Rb, Rb-Ser(P)-807/811, Rb-Ser(P)-795, Cdk2, Cdk4, CyclinD1, GFP, Thr(P), perifosine, and human recombinant IGF-1 were from Cell Signaling Technology. Anti-MCM7, Flag, and V5 antibodies were purchased from Santa Cruz. Antibody for RACK1 was from BD Transduction Laboratories. Antibodies for MCM4, MCM6, Cdt1, p27, E2F1, and Histone 3 were from Proteintech Group. Anti-Ser(P) antibody was from Millipore.
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3

Western Blot Analysis of Cellular Signaling

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Cells were lysed and subjected to standard western blotting.30 The primary antibodies against EZH2 (#5246), p110α (#4249), pAKT‐Ser473 (#4060), AKT (#4691), pRB‐Ser780 (#3590), pRB‐Ser807/811 (#8516), RB (#9313), H3K27me3 (#9733), p21 (#2947) (Cell Signaling Technology, Danvers, MA, USA), Histone3 (#17168‐11‐AP), GAPDH (#60004‐1‐1G) (Proteintech Group, Chicago, IL, USA) and β‐actin (#A5441) (Sigma–Aldrich) were used at the recommended dilutions. Images were captured by a ChemiDoc Touch Imaging System (Bio‐Rad).
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4

Western Blot Analysis of Epigenetic Regulators

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Cells and tissues were lysed in RIPA (P0013B, Beyotime, China) supplemented with 1 mM PMSF (ST506, Beyotime, China) and 0.01 mM phosphatase inhibitors (P1260, Solarbio, China). Samples with 20 ug protein in each group were separated by 10% SDS-PAGE and electrotransferred to PVDF membranes (Millipore, USA). The primary antibodies were used as following: β-actin (1:2000, 20536-1-AP, Protein tech, China), EZH2 (1:1000, #5246, Cell Signaling Technology, USA), SUZ12 (1:1000, #3737, Cell signaling Technology, USA), EED (1:1000, 16818-1-AP, Protein tech, China), Histone 3 (1:2000, 17168-1-AP Protein tech, China), H3K27me3 (1:1000, #9733, Cell Signaling Technology, USA), and H3K27ac (1:1000, #8173, Cell Signaling Technology, USA). After incubated with horseradish peroxidase-conjugated secondary antibody (1:2000, #7074, Cell Signaling Technology, USA), the blots were visualized with chemiluminescent substrate (US Everbright Inc., China) by Image Lab (Bio-Rad, USA).
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5

Immunoblotting analysis of mitochondrial and DNA repair proteins

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Primary antibodies against NDUFS1 (no. 12444), NDUFA9 (no. 20312-1-AP), NDUFV1 (no. 11238), NDUFV2 (no. 5301), KU80 (no. 6389), KU70 (no. 10723), RAD51 (no. 14961), PARP-1 (no. 13371), PDP1 (no. 21176),PDK1 (no. 10026), β-actin (no. 60008), and Histone3 (no.17168) were obtained from Proteintech and γ-H2AX (no. AP0099), PDHA1 (no. A17432) were obtained from Abclonal and p-PDH (no.ab115343), HIF1a (no. ab179483) were obtained from Abcam, and Acetyl-Histone H3 (Lys9) (no. 9649 s), Acetyl-Histone H3 (Lys56) (no. 4243 s), were obtained from CST in this study.
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6

LPS-induced BMDM protein analysis

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After being treated with LPS, the total protein level of the BMDMs was collected using RIPA (Beyotime, China) and protease inhibitor (Roche, USA). Additionally, the cytoplasmic and nuclear protein fractions were extracted by using the ProteinExt Mammalian Nuclear and Cytoplasmic Protein Extraction Kit (TransGen Biotech, China). The levels of TRIM59 (Abcam, USA), p65 (CST, USA), p-p65 (CST, USA), IκB (CST, USA), p- IκB (CST, USA), IKKα (CST, USA), IKKβ (CST, USA), p-IKKα/β (CST, USA), ECSIT (ABclonal Biotechnology, China), MAP3K1 (ABclonal Biotechnology, China), AKT (CST, USA), p-AKTSer473 (CST, USA), p-AKTThr308 (CST, USA), PI3K (CST, USA), p-PI3K (CST, USA), GAPDH (Proteintech, China), and Histone-3 (H3, Proteintech, China) were measured using Western blot analysis.
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