Fig. 1

Induction of survival motor neuron gene (SMN) expression in interferon (IFN)-treated cells. (A) HeLa cell proteins (50 µg) were solubilized and run on a 12% polyacrylamide gel and blotted. The Western blot was cut into two strips and each strip was probed with (a) the preimmune serum or (b) the immune serum (739785) at a dilution 1:2000. (B) 24 hr after subculture, exponentially growing A172 and HOG cells were treated with or control without 1000 U/ml of IFN-β or 1000 U/ml of IFN-γ. After 16 hr of culture, cells were lysed and total protein extracts (50 µg) were analyzed by Western blots analysis, using the polyclonal antibody against SMN (739785) (1:2000) followed by horseradish (HRP)-labeled conjugate antibody at a dilution 1:2000. Immunodetection was realized by enhanced chemiluminescent protein (ECL) reagents and autoradiography. The blot was rehybridized with a monoclonal anti-actin antibody to ensure that equal amount of proteins were present in each lane. (C) Total cell extracts from untreated (C) or treated A172 cells for 8, 16 and 24 hr with IFN-β (1000 U/ml) or IFN-γ (1000 U/ml) were analyzed by Western blots analysis, using anti-SMN and anti-actin antibodies as described in (B). (D) A172 cells were cultured for the indicated time periods or control with medium alone (C) in the presence of IFN-β (1000 U/ml) or IFN-γ (1000 U/ml). Total cellular RNA (20 pg/lane) was subjected to agarose gel electrophoresis, transferred to nylon membrane, and hybridized with a (³²P)-labeled human SMN cDNA probe. Filters were subsequently hybridized to a 28S rRNA oligonucleotide probe as a control of RNA loading.