Anti cyclin e1

Ham’s F-12K medium, McCoy’s 5A medium, Dulbecco’s modified Eagle’s medium (DMEM), Minimum Essential Medium (MEM), 100 U/mL streptomycin, and 100 μg/mL penicillin were acquired from Gibco (Life Technologies, Waltham, MA, USA). Fetal bovine serum was obtained from BI (Biological Industries, Beit Haemek, Israel). An SRB assay kit was purchased from Bestbio (Shanghai, China). Cell cycle and apoptosis analysis kits were purchased from Yeasen Biotech (Shanghai, China). A Reactive Oxygen Species Assay Kit, cycloheximide (CHX), and Hoechst 33258 were obtained from Beyotime (Beyotime Biotechnology, Shanghai, China). Normal goat serum was purchased from Boster (Boster Biological Technology, Wuhan, China). Anti-Cyclin D1, anti-Cyclin E1, anti-P27, anti-GAPDH, anti-β-actin, anti-FOXO3A, anti-SKP2, anti-γH2AX, HRP-conjugated affinipure goat anti-rabbit, HRP-conjugated affinipure goat anti-mouse, and CoraLite488-conjugated affinipure goat anti-rabbit were purchased from Protein Tech (Proteintech group, Wuhan, China). Ultrapure water was produced with an ultrapure water system (Yipu Yida Technology, Nanjing, China). All of the analytically pure chemicals and solvents were purchased from Sinopharm Group (Shanghai, China).

Virus-infected or mock-infected cells were collected at various times after EV-D68 infection and washed once with PBS as previously described (Yu et al.). The following antibodies were used in Western blot analyses: anti-CDK2 (Cell Signal), anti-cyclinE1 (Proteintech), anti-CDK4 (Cell Signal), anti-CDK6 (Cell Signal), anti-cyclinD (Cell Signal), anti-CDK1 (Boster), anti-cyclinB1 (Santa Cruz), and anti-histone (GenScript). Secondary antibodies from mouse or rabbit were obtained from Jackson Immuno Research.

Total protein samples were extracted from tissues and cultured cells. Then, the samples were separated using 10–12% SDS-PAGE and transferred onto a polyvinylidene fluoride membrane. After blocking with 5% non-fat milk in Tri-buffered saline for 1.5 h, the membranes were incubated at 4°C overnight with target antibodies against the following proteins: anti-SHMT2 (1:1,000, 11099-1-AP; Proteintech, Wuhan, China), anti-GAPDH (1:10,000; 60004-1-Ig; Proteintech, Wuhan, China),anti-Cleaved-caspase 3 (1:1,000, #9661S; CST, United States), anti-Cyclin D1 (1:1,000, 60186-1-Ig; Proteintech, Wuhan, China), anti-Cyclin E1 (1:1,000, 11554-1-AP; Proteintech, Wuhan, China), anti-Bcl-2 (1:1,000, #2872T; CST, United States), anti-Bax (1:1,000, 50599-2-Ig; Proteintech, Wuhan, China), anti-NF-κB P65 (1:1,000, #8242T; CST, United States), anti-p-NF-κB P65 (Ser536) (1:1,000, #3033; CST, United States), anti-STAT3 (1:1,000, #9139; CST, United States), anti-p-STAT3 (Tyr705) (1:2000, #9145; CST, United States). GAPDH was used as an internal loading control. After washing three times with TBST, the membranes were incubated with species-special secondary antibodies at room temperature for 1 h. Next, the membranes were detected by the ChemiDoc^TM Touch Imaging System (Bio-Rad, Hercules, United States). Finally, relative protein expression levels were assessed using the image J software.