Actin

The cells were lysed for 30 min and then centrifuged at 13,000× g for 30 min at 4 °C. The quantified protein was mixed in proportion with the protein loading buffer and boiled at 95 °C for 10 min. The sample was electrophoresed and then transferred to a polyvinylidene difluoride (PVDF) membrane. The skim milk powder was diluted with Tween-20 Tris buffered saline (TBST) for 1 h at room temperature, then incubated with the primary antibody overnight, washed with TBST, and then incubated with the corresponding secondary antibody for 1 h at room temperature. The reaction was visualized using electrochemiluminescence (ECL, Bio-Rad, Hercules, CA, USA) and detected by exposure to autoradiographic film. The densitometric reading/intensity ratio for each strip was included in all western blots (Figures S3 and S4).
The primary antibodies used included CyclinD1 from BD Pharmingen(SanDiego, CA, USA); p-IĸB, p-NFĸB1, Bcl-2, Bax, p-TAK1, p-IKK, caspase3, and cleaved caspase3 from Cell Signaling Technology(Danvers, MA, USA); actin from Transgen Biotech(Beijing, China); C20orf27 and p-TGFβR1 from Abcam(Cambridge, MA, USA); Bay11-7082 from TargetMol(Boston, MA, USA); and PP1c, GADD34, and TAK1 from Santa Crus (CA, USA).