Mouse anti gfp mab

mts4-ts cells expressing Cnp3-GFP were grown in YES to midlog phase at 25ºC and then shifted to 36ºC for 3 h. 45 min before harvesting, 5 mM N-ethylmaleimide (E3876; Sigma-Aldrich) was added to the culture media to inhibit deubiquitinating enzymes. Cells were harvested by centrifugation, and the pellets were washed with ice-cold STOP buffer and snap-frozen in liquid nitrogen. Native extracts were prepared in PBS with protease inhibitor cocktail (8340; Sigma-Aldrich) and 10 mM PMSF. 6 mg of total protein extracts was incubated with anti–mouse IgG–coated magnetic beads (Dynabeads; 11201D; Thermo Fisher) for 1 h at 4ºC. Beads were washed three times with PBS with protease inhibitors, resuspended in 50 µl SDS-PAGE sample loading buffer (0.25 M Tris-HCl, pH 6.8, 8% [wt/vol] SDS, 0.004% [wt/vol] bromophenol blue, and 20% [vol/vol] 2-mercaptoethanol), and boiled for 5 min. Samples were clarified by centrifugation at 13,000 rpm for 10 min, and 20 µl was immunoblotted with mouse anti-GFP mAb (11814460001; Roche) to detect Cnp3-GFP and rabbit antiubiquitin pAb (sc-9133; Santa Cruz; provided by J.A. Sanchez Alcazar, CABD/UPO) to detect ubiquitinated proteins.

For the analysis of the half-life of Cnp3 and Cdc13, WT and alm1Δ cells were grown in YES at 25ºC to midlog phase, and then 100 µg/ml CHX (01810; Sigma-Aldrich) was added to the cultures. Cells were harvested at the indicated time points, and whole cell extracts were prepared for immunoblotting using mouse anti-GFP mAb (11814460001; Roche) to detect Cnp3-GFP, rabbit anti-Cdc13 pAb (SP4, provided by S. Moreno), and mouse anti-PSTAIR mAb (P7962; Sigma-Aldrich) as a loading control. ImageJ software (version 1.5; National Institutes of Health) was used to quantify Cnp3-GFP and Cdc13 protein levels.