Fig. 2
MG132 upregulated the level of EGFP-tagged C protein. a PK-15 cells were transfected with plasmid pEGFP-N1-C followed by treatment of 10 μM MG132 or 5 mM 3-MA or both of them for 16 h. DMSO or ddH2O were used as controls. At 20 hpt, cells were lysed and subjected to Western blot analysis with the indicated antibodies. Tubulin was used as a control. b Schematic representation of EGFP-C protein and its cleaved form. Black frame represents C protein and green frame represents EGFP protein. The arrow indicates the cleavage site of C protein by SPP. PK-15 cells were transfected with plasmid pEGFP-C1-C and were treated with or without MG132 (10 μM). Empty vector pEGFP-C1 was used as a control. Cells were lysed at 20 hpt and Western blot was performed with the indicated antibodies. c PK-15 cells were transfected with plasmid pEGFP-N1-C and treated with or without MG132 as described above. Empty vector pEGFP-N1 was used as a control. Western blot was performed as described above. Relative mRNA levels of EGFP (d) and C (e) in cells were detected by qRT-PCR. Data was analyzed by Student’s t-test. f 3D4/2 cells were transfected with plasmid pEGFP-N1-C or pEGFP-N1 followed by treatment of 10 μM MG132 or same volume of DMSO for 16 h. The fluorescence in cells was observed at 20 hpt. Scale bar, 100 μm. g Cells in (f) were then lysed and analysed by Western blot with the indicated antibodies. Protein marker is shown on the right. Data in all bar plots are shown as mean ± SD and representative of 3 biological replicates. ***, P< 0.001; ns, not significant