The capacity to remove toxic proteins that accumulate in neurodegenerative disorders attests to the disease-modifying potential of the autophagy pathway. Coprecipitated proteins were resolved on SDS ͞ PAGE and detected by Western blot analysis by using anti-His antibody.Īutophagy is a conserved, multi-step process of capturing proteolytic cargo in autophagosomes for lysosome degradation. GST or GST-MTMR5 was pulled down by glutathione-agarose beads. As a control, equimolar GST was incubated with His-tagged MTMR2. ( D ) Puri fi ed GST-tagged bacterial recombinant MTMR5 (10 g) was incubated with equimolar His-tagged MTM1 or MTMR2 fusion proteins. Thirty hours after transfection, cell extracts were subjected to immunoprecipitation by using anti-FLAG antibody, and endogenous MTMR5 was detected by Western blot analysis by using anti-MTMR5 antibody. ( C ) 293 cells were transfected with FLAG- MTM1 or FLAG -MTMR2 expression vectors. The peptide masses analyzed by MALDI ͞ post source decay are underlined. Shown in bold are peaks unique to the p200 band compared with control samples, which correspond to matched peptides to human MTMR5. ( B ) MALDI ͞ TOF mass spectrometry of the typtic digests of MTMR5. The p200 band excised for MALDI ͞ TOF mass spectrometry analysis is marked with an arrow. Immunoprecipitated proteins were separated by SDS ͞ PAGE, and the gel was stained with Coomassie blue. Thirty hours after transfection, cell extracts were subjected to immunoprecipitation by using anti-FLAG antibody. ( A ) 293 cells were transfected with either FLAG- MTM1 or FLAG- MTMR2 expression vectors. Identi fi cation of an MTMR2-interacting protein.
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