Fig 1: Heat shock-induced SUMOylation is also reduced in fugu5.(A) Phenotypic analysis of 10-days-old Col-0, fugu5-1, UBQ:PPa5-GFP in Col-0 and fugu5-1 backgrounds and UBQ:AVP1 seedlings analysis before and after heat. Representative pictures of seedlings before and 4 days after completion of heat shock treatment (40°C, 4 hours) are depicted. (B) Seedling survival was determined 4 days after the heat shock. Alive and dead seedlings were counted and survival is shown as the percentage of the living seedlings. Error bars show SD of the mean with n = 24 samples of one representative experiment. Two biological experiments were performed. Asterisk indicates significant difference compared to Col-0 (Student’s t test; **p<0.01, ***p<0.001). (C) SUMOylation levels of Col-0 and V-PPase mutant fugu5-1 were analysed with western blot under normal conditions (22°C) and after heat shock treatment (40°C, 30 min). (D) Measurement of the SUMO amount of Col-0, V-PPase mutants, UBQ:PPa5-GFP/fugu5-1 and UBQ:AVP1 seedlings after heat shock treatment (40°C, 30 min). (C) and (D) 10-days-old liquid grown seedlings are used for total protein extraction. Anti-SUMO1/2 (Agrisera) was used as primary antibody. Whole lanes were measured for the calculation of protein amounts using ImageJ. cFBPase detection was used for normalization. Error bars represent SD of n = 2 biological replicates. Asterisk indicates significant difference compared to Col-0 (Student’s t test; *p<0.05, **p<0.01, ***p<0.001).10.7554/eLife.44213.015Figure 4—source data 1.(B) Survival measurement of the 10 days old seedlings upon heat shock.(C-D) Comparison of the amount of the total SUMOylation with and without heat shock treatment.