The Growth and Tumor Suppressors NORE1A and RASSF1A Are Targets for Calpain-Mediated Proteolysis
Figure 4
A, The addition of a calpain inhibitor ALLN to cultured cells prevents NORE1A degradation: NORE1A was re-expressed in H157 cells (lanes 1, 6), H358 cells (lanes 3, 8) and H460 cells (lanes 5, 10) using a vector with a drug-resistance marker with subsequent selection for the drug. Lanes 2, 7 represent H358 parental cells and lanes 4, 9 - H460 parental cells. Cells were cultured for 3 days without inhibitors (top panel) or with 1 µM ALLN (bottom panel). Cell extracts, equalized by α-tubulin, were probed with 10F10 antibody by Western Blotting. At 1 µM concentration ALLN did not induce apoptotic response in H358 and H460 cells (data not shown). B, NORE1A expression at the mRNA level in H358 and H460 cells: NORE1A cDNA was re-expressed in H358 and H460 cells using a vector with a drug-resistance marker. Total mRNA was extracted from pools of drug-resistant cells emerged after selection as indicated and analyzed by RT-PCR for NORE1A expression. Note that these cells did not express NORE1A protein (Fig 4A, lanes 3 and 5). NORE1A, cDNA of vector used for transduction was included as a positive control. M, DNA molecular weight markers. C: Extracts of human tumors are capable of degrading NORE1A. Human lung tumors from patients 3, 9, 10 and 8 and matching normal lung tissues were extracted into buffer for cell extraction and normalized by total amount of cellular protein. Equal amount of extracts from tumors (T) and normal tissues (N) were mixed with FLAG-tagged NORE1A, immunopurified as described in Methods. After thirty minute incubation at 37°C, electrophoretic sample buffer was added to 1× concentration, samples were boiled and subjected to Western blotting with anti-FLAG antibodies. As positive control for NORE1A degradation, NORE1A incubated with H460 cell extract (lane 2) was used.