Primary cardiac myocytes were cultured under normoxic conditions for 48 h following isolation, and the media was then replaced with ischemic buffer and the cells subjected to four hours of simulated ischemia and 24 h of reperfusion. Mitochondria were stained with Mitotracker-Red (Molecular Probes) and the cells fixed with 4% (w/v) paraformaldehyde for 15 min then stained with anti-STAT1 or anti-STAT3 antibodies. As shown in Figure 1A, STAT1 was found to form punctate staining where STAT1 co-localized with mitochondria. We the autophagosome membrane and shares 28% homology with yeast Apg8/Aug7p which has been shown to be important in yeast autophagosome formation. 26,27 Studies have shown that during I/R injury, autophagy is activated and is thought to promote cell survival and reduce infarct size. Studies from our laboratory have shown that STAT1, as well as promoting apoptosis, negatively regulates autophagy. 19 We propose that by enhancing apoptosis and negatively regulating autophagy, STAT1 promotes the generation of larger infarcts which can lead to heart failure. Recently, another catabolic cellular process, mitophagy, has received much attention. Figure 1. primary cardiomyocytes were seeded onto gelatin coated-glass coverslips and subjected to normoxic (control) conditions or simulated ischemia/reperfusion injury, stained with 25 nM Mitotracker-Red then fixed with 4% paraformaldehyde. Immunofluorescent staining indicated that STAT1 co-localized to the mitochondria before I/R injury but formed more localized, punctate staining following I/R injury (A) while STAT3, formed localized punctate staining both before and after I/R injury (B). In order to confirm the presence of STAT1 in the mitochondrial fraction, subcellular fractionation of primary cardiac fibroblasts under normoxic conditions was performed and the mitochondrial (M), cytosolic (c) and nuclear (N) fractions run on 10% pAGe gels and western blotting indicated the presence of STAT1 in all three fractions (C). In order to ensure that the presence of STAT1 was not due to contamination, the blots were interrogated for the cytosolic marker, GApDh and the mitochondrial membrane marker, coxIV (C). injury as described above and the mitochondrial and cytosolic fractions isolated. Western blotting of these fractions and interrogated for STAT1 and STAT3 revealed that both STAT1 and STAT3 appeared to move from the mitochondrial fraction into the cytosolic fraction following simulated I/R injury (Fig. 3C).

### Language ###
English

### Assessment ###
We can ask about the cellular location of STAT1 under normal conditions, as this is clearly stated in the text.

### Query ###
Where is STAT1 protein found inside cells under normal conditions?
Where is STAT1 protein found inside cells under normal conditions?