Anti β tubulin

Primary mouse anti-Oct4, anti-Sox2, and anti-Klf4 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA); rabbit anti-mono-H3K27 from Abcam; rabbit anti-mono-,di-,tri-H3K4, rabbit anti-di-,tri-H3K27, and rabbit anti-Nanog from Cell Signaling Technology (Danvers, MA, USA); rabbit anti-Nestin from Sigma (Sigma, St. Louis, MO, USA); and anti-β-tubulin and anti-β-actin from Beyotime Institute of Biotechnology (Jiangsu, China). miRNA inhibitors were purchased from Biomics Biotechnologies (Jiangsu, China).

Western blotting was performed as described (58 (link)). The following primary antibodies were used: mouse anti-V5 (1:5,000; Thermo Fisher Scientific, Waltham, MA, USA), mouse anti-tubulin (1:2,000; Solarbio, Beijing, China), rabbit Anti-GAPDH (1:2,000; Solarbio), and rabbit anti-Hypusine (EMD Millipore; 1:1,000) antibodies, rabbit anti-eIF5A (1:1,000; ABclonal), rabbit anti-DHPS (1:200; abcam), rabbit anti-DOHH (1:500; Sigma-Aldrich). Secondary antibodies used were Goat Anti-Mouse horseradish peroxidase-conjugated secondary antibody (1:2,000; Beyotime, Shanghai, China) and Goat Anti-Rabbit horseradish peroxidase-conjugated secondary antibody (1:5,000). Anti-His (M1001020, Solarbio), Anti-pAKT473 (GB13012-3, Servicebio), Anti-pAKT308 (ab66134, Abcam), Anti-ATPaseβ (GL Binchem synthesis), Anti-Inx1(GL Binchem synthesis), Anti-Inx2 (GL Binchem synthesis), Anti-Inx3(GL Binchem synthesis), Anti-Inx4 (GL Binchem synthesis), Cleaved-caspase (WL02117, Wanleibio), Anti-GAPDH (M1000110, Solarbio), Anti-β Tubulin (AF1216, Beyotime), Goat Anti-Mouse (A0216, Beyotime), Goat Anti-Rabbit (A0208, Beyotime). Proteins were semi-quantified via densitometry using ImageJ (National Institutes of Health, Bethesda, MD, USA).

The total proteins of both cell lines were extracted and measured by Western blot analysis with the following antibodies: anti-H3K9me3 (Abcam, MA, USA; 1:1000), anti-HP1α (Abcam, MA, USA; 1:2000), anti-Ac-H3 (CST, 1:1000) and anti-β-Tubulin (Beyotime Biotechnology, 1:1000). The cells were washed triply with ice-cold PBS and treated with RIPA lysis buffer with 100 mM phenylmethanesulfonyl fluoride (PMSF) (Beyotime). An equal amount of total protein was subjected to 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, Bedford, MA, USA) then probed with primary antibodies overnight at 4°C. The membranes were then incubated for 1 h with the secondary HRP-conjugated antibodies (1:1000; Cell Signaling Technology). β-Tubulin was used for the loading control. The protein bands were visualized using the ChemiDoc XRS system (Bio-Rad Laboratories, Hercules, CA, USA) and their densities were measured using the Quantity One software (Bio-Rad Laboratories).