MECA is destroyed by the proteasome and autophagy. (A and B) Proteasome inhibition during meiosis by treatment with MG-132, compared with a DMSO vehicle control. Flasks were split at 5 h, with one half treated with 100 µM MG-132 and the other half with a DMSO vehicle control. Band intensity quantifications, normalized to the Hxk2 loading control, and time course staging by DAPI staining for each time point are shown below the immunoblots. (A) Immunoblot of Num1-3V5 in a pGAL-NDT80 GAL4.ER synchronous meiosis (UB13245), where 1 µM β-estradiol was added to the flasks at 5 h. Num1 runs above the highest (190-kD) ladder band. (B) Immunoblot of Mdm36-3V5 (UB16324). Asterisk: band of unknown identity. (C) Immunoblot autophagy assay of GFP-Mdm36 (UB16326). (D) Quantification of the blot in C. The total level of full-length protein (normalized Mdm36/Hxk2) was calculated as the intensity of the GFP-Mdm36 band divided by the Hxk2 loading control band. Percentages of the maximum value are plotted. The percentage free GFP value was calculated as the intensity of the free GFP band divided by the summed intensities of the free GFP and GFP-Mdm36 bands.