Figure 9. Immunoblot analysis of tissue extracts from pterygia and normal conjunctiva. Examination of hematoxylin and eosin-stained frozen sections from frozen tissue blocks was performed before the western blotting. A: The extracts probed with anti-HIF-1α (first panel), anti-Hsp90 (second panel), anti-Hsp70 (third panel), anti-pVHL (fourth panel), anti-phosphoHsp27 (fifth panel), anti-Hsp27 (sixth panel), and antibody against tubulin (bottom panel). The same amount of protein (50 μg) was loaded into each lane. Positions of molecular mass markers (in kilodaltons) are indicated on the left. The HIF-1α at 120 kDa was detected only in pterygia. Multiple bands of weaker intensity at 100–120 kDa represent post-translational modification of HIF-1α. The 90 kDa Hsp90 protein was expressed in both pterygium and normal conjunctiva samples. A 70 kDa band for Hsp70 was found in both pterygium and normal conjunctiva samples. Molecular weight of Hsp27 is 27 kDa, and its phosphorylated isoform phospho-HSP27 has been noted to be about 27 kDa. Hsp27 and phospho-Hsp27 were expressed in both pterygium and normal conjunctiva samples. For pVHL, the major band detected around the 30 kDa and one additional band of weaker intensity at 14-KDa were also noted (not shown). The pVHL-immunoreactive band was hardly detectable in pterygia, and similar pVHL-immunoreactive bands were observed for normal conjunctival samples. B: The corresponding densitometry analysis. The higher protein expression in pterygia compared to normal conjunctiva was statistically significant for Hsp90 (***p<0.00001), Hsp70 (***p<0.00001), and Hsp27 and phospho-Hsp27 (***p = 0.0001). No significant difference was detected for pVHL expression (p = 0.3). No statistical analysis was performed for HIF-1α expression since all pterygium samples demonstrated HIF-1α expression whereas HIF-1α was not detected in normal conjunctival samples.