Figure 2.

Social isolation induces the UPR. BiP expression is increased in flies that have been socially isolated (SI) in comparison to the grouped/socially enriched (SE) flies. SE flies were placed in vials and housed in groups of 20–30 flies from eclosion until their behavior was recorded. SI flies were isolated from pupae, sexed, individually housed in 2 ml eppendorf tubes and behavior was recorded 5–7 days later. (a) Densitometric quantification and (b) representative western blot of BiP expression in wCS10 fly heads (n = 4 trials, 10 heads/pool). Blot shows 10 µg of protein loaded per well from pooled socially enriched (SE) and socially isolated (SI) lysate; fold change (mean + standard error) in BiP expression in the SI flies over grouped p = .04. Dual lanes represent technical replicates. (c) xbp1 mRNA splicing is increased in SI flies compared to the grouped/SE flies. Splicing was measured by reverse transcription polymerase chain reaction (RT-PCR). Whole flies where used for these experiments (n = 24/condition; 2 flies/sample). Histogram represents data averaged from 3 PCR reactions and gels representing 12 independent samples per condition. Spliced and unspliced xbp1 were quantified from gels using Biorad image software and normalized across gels to the average of grouped xbp1. (d) Representative gel showing xbp1s and xbp1u; lanes represent independent biological replicates. Although xbp1 was found to be spliced in both grouped (SE) and socially isolated (SI) flies, more of both the unspliced and spliced variants were found under SI conditions. (e) p-eIF2α levels are increased in the SI flies compared to the grouped/SE flies (n = 10 per group, p = .02). (f and g) The effect of SI on BiP levels is reversible. Flies were grouped/SE, SI, or raised in SI then grouped for three (3) days. Placing SI flies in a grouped/SE environment both increased their sleep (data not shown) and reduced BiP levels (p < .05). Mean ± SEM shown, *p < .05, **p < .01, ***p < .001.