FIGURE 3.

SNON mRNA and protein levels are regulated at different levels after TGF-β stimulus. A, A549 cells were incubated for different times with or without 300 pm TGF-β. Total RNA was isolated, and then SNON (308-bp), SMAD7 (317-bp), and β-actin (237-bp) mRNAs were amplified by RT-PCR with specific primers (n = 3). B, A549 cells were incubated for different times in the presence or absence of 300 pm TGF-β, and in the indicated cases, 5 μg/ml actinomycin D (ACD) or 10 μm SB431542 (SB43) was added 2 h post-treatment with TGF-β. Total RNA was isolated, and RT-PCR was performed with specific primers for SNON (308 bp), SMAD7 (317 bp), and β-actin (237 bp) (n = 2). C and D, A549 cells were preincubated for 30 min in the presence or absence of 10 μm SB431542 and then incubated for 1 h with or without 300 pm TGF-β. Total RNA was isolated, and RT-PCR was performed with specific primers for SNON and β-actin (n = 2) (C), or whole cell protein extracts were immunoprecipitated with anti-SNON or anti-S2/S3 antibody and then detected by WB with anti-SNON or anti-phospho-S2 antibody (n = 3) (D).