PMC

A. Representative sumo-2/3 immunoblot from 30 minute OGD/hypothermia experiment. Control cultures (Con) were harvested 24 hours after treatment. Hypothermia cultures (H) were harvested 1 hour after treatment, while 30 minute OGD cultures were harvested at 1, 4, 8 and 24 hours. B. Representative sumo-2/3 immunoblot from 120 minute OGD/hypothermia experiment. Control cultures (Con) were harvested 24 hours after treatment. Hypothermia cultures (H) were harvested 1 hour after treatment, while 120 minute OGD cultures were harvested at 1, 4, 8 and 24 hours. C. Quantification of sumo-2/3 intensities of the 85+ kDa region from 30 and 120 minute OGD experiments. Normalization was carried out against the intensity of controls, the error bars are standard deviation, *** P<0.001, two-way ANOVA with post-hoc Bonferroni compared to control. D. Representative sumo-1 immunoblot from 120 minute OGD/hypothermia experiment. Control cultures (Con) were harvested 24 hours after treatment. Hypothermia cultures (H) were harvested 1 hour after treatment, while 120 minute OGD cultures were harvested at 1, 4, 8 and 24 hours. E. Quantification of sumo-1 intensities of the 85+ kDa region from 120 minute OGD experiments (* denotes P<0.05). All data are the result of 4 to 5 independent experiments.

A. Representative sumo-2/3 immunoblot from cycloheximide/ischemic preconditioning experiment. Cycloheximide group: Control cultures and cultures subjected to 120 minute OGD were maintained in media containing 1 µM cycloheximide for 24 hours prior to treatment. Preconditioned cultures were subjected to 30 minute OGD and maintained in media with 1 µM cycloheximide for 24 hours prior to 120 minute OGD. All cultures were harvested 1 hour after treatment. No cycloheximide group: Identical procedures, but without cycloheximide. B. Quantification of sumo-2/3 intensities of the 85+ kDa region after cycloheximide/preconditioning experiments. All cultures harvested 1 hour after treatments. Normalization was carried out against control values, the error bars are standard deviation. * P<0.05, *** P<0.001 compared to control, # P<0.05 compared to 120 without cycloheximide, ++P<0.01 compared to 30, 120 without cycloheximide, oneway ANOVA with post-hoc Bonferroni’s test. All data are the result of 4 to 5 independent experiments.

A. Representative sumo-2/3 immunoblot from ischemic preconditioning experiment. Control cultures and cultures subjected to 30 or 120 minute OGD were harvested 1 hour after treatment. Preconditioned cultures were subjected to 30 minute OGD 24 hours prior to 120 minute OGD and harvested 1 hour later. B. Representative sumo-2/3 immunoblot from hypothermic preconditioning experiment. (The dotted lines are indicative of the fact that this image is a rearranged composite created from a single immunoblot, for reasons of clarity and correspondence with . Any adjustments to brightness or contrast were performed on the entire image prior to clipping.) Control cultures and cultures subjected to 120 minute OGD or 30 minute hypothermia were harvested 1 hour after treatment. Preconditioned cultures were subjected to 30 minute hypothermia 24 hours prior to 120 minute OGD and harvested 1 hour later. C. Upper panel, quantification of sumo-2/3 intensities of the 85+ kDa region after preconditioning experiments. All cultures harvested 1 hour after treatments. Lower panel, LDH release 24 hours after preconditioning experiments. Normalization was carried out against control values, the error bars are standard deviation. ** P<0.01, *** P<0.001 compared to control, # P<0.05, ## P<0.01 compared to 120 minute OGD, one-way ANOVA with post hoc Bonferroni’s test. D. Upper panel, quantification of sumo-2/3 intensities of the 85+ kDa region after preconditioning experiments. All cultures harvested 1 hour after treatments. Lower panel, LDH release 24 hours after preconditioning experiments. Normalization was carried out against control values, the error bars are standard deviation. ** P<0.01, *** P<0.001 compared to control, ## P<0.01, ### P<0.001 compared to 120 minute OGD, one-way ANOVA with post-hoc Bonferroni’s test. All data are the result of 4 to 5 independent experiments.

A. Representative sumo-2/3 immunoblot from OGD/recovery time course experiment (non-heat-treated tissue). Control cultures were harvested 60 minutes after treatment. OGD cultures were harvested after 30, 60, 90, or 120 minute OGD. Recovery cultures were replenished with Neurobasal A medium at the end of 120 minute OGD and harvested 30 or 60 minutes later. B. Quantification of sumo-2/3 intensities of the 85+ kDa region from OGD/recovery time course experiments. Normalization was carried out against the intensity of controls, the error bars are standard deviation, * P<0.05, **P<0.01, one-way ANOVA post hoc with Dunnet’s test compared to control. All data are the result of 4 to 5 independent experiments. C. Representative Ubc9 and UBA2 immunoblots from ischemic preconditioning experiment. Control cultures and cultures subjected to 30 minute preconditioning OGD were harvested 24 hours after treatment. Alpha tubulin is shown as a loading control. D. Representative SenP1, SenP2 and SenP3 immunoblots from ischemic preconditioning experiment. Control cultures and cultures subjected to 30 minute preconditioning OGD were harvested 24 hours after treatment. E. Quantification of Ubc9 levels and UBA2 levels from ischemic preconditioning experiment. Normalization was carried out against the intensity of controls, the error bars are standard deviation, data were tested for significance using two-way ANOVA with post hoc Bonferroni’s test compared to control. All data are the result of 3 or 4 independent experiments. F. Quantification of SenP1, SenP2 and SenP3 levels from ischemic preconditioning experiment. Normalization was carried out against the intensity of controls, the error bars are standard deviation. Data were tested for significance using two-way ANOVA with post hoc Bonferroni’s test compared to control. All data are the result of 3 independent experiments.