Templates for splicing substrates were generated by digestion of pEZ_ISS/WT with Nar I. Labeled RNA was synthesized from linear plasmid in a 50 µl reaction containing 2 µg of DNA, 0.4 mM each of ATP and GTP, 0.1 mM UTP and CTP, 2.5 µl of [32P]-UTP and [32P]-CTP (ICN), 0.5 mM diguanosine triphosphate and 2 µl of 20 U/µl T7 RNA polymerase Plus (Ambion, Austin, TX).
The intracellular domain of the NOTCH1 (ICN) plasmid pSIN-EF2-ICN was kindly provided by Prof. Lei Dong (State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University).
In contrast, the ectopic overexpression of AMFR and NOTCH1 using plasmids of AMFR and NOTCH1 (ICN) ORF promoted cell invasion, which could not be repressed by the overexpression of miR-139-5p miR-139-5p miR-139-5p
After Zymolyase treatment (1 mg/ml Zymolyase 100T ICN) for 30 min at 37°C, cells were washed and stored at 4°C for antibody staining.
BCKA used in this assay were: α-Ketoisocaproic acid sodium salt (α-KIC; K0629, SIGMA), DL-α-Keto-β-methylvaleric acid sodium salt (α-KMV; K7125, SIGMA) and Sodium methyl valerate (α-KIV; 151395, ICN).
They were washed in PBST, blocked in 10% bovine calf serum (BCS), and incubated with primary antibody overnight at 4°C (rabbit anti-β-galactosidase, 1∶10000, ICN) (mouse anti-Gro, 1∶100, kind gift from Christos Delidakis).
The remainder of the cells were washed three times in 10 ml of ice cold 10 mM Tris pH 7.5, 50 mM EDTA pH 8 and resuspended in 110 µl of 10 mM Tris pH 7.5, 500 mM EDTA pH 8 containing 2.5 mg/ml zymolyase-100T (ICN).
Briefly, S. cerevisiae THY.AP5 cells expressing different HMT-1-NubG-fused constructs, or the empty NubG vector were converted to spheroplasts with Zymolyase 20T (ICN) in a buffer that also contained 1% (w/v) yeast extract, 2% (w/v) Bacto-Peptone) 0.7 M sorbitol, 1% (w/v) dextrose, 5 mM dithiothreitol, and 100 mM Tris-Mes (pH 7.5).
The concentrations used were based on those reported: IFN-γ (100 U ml−1) (Sigma); TNF-α 10 ng ml−1 (ICN); IL-1β 10 U ml−1 (Sigma); and IL-4 100 U ml−1 (Sigma).
Actin blots to ensure equivalent loading were developed with clone C4 (ICN) diluted at 1 80,000.
