Fig 6. Effects of HSC70 on HIF-1alpha regulation by PDI.

(A) Hep3B cells overexpressing PDI or HSC70 were cultured in the presence of 10 mM NH₄Cl or 10 μM leupeptin for 8 h under hypoxic conditions. Proteins (3 μg) were separated by SDS-PAGE, and immunoblotting was performed with the anti-HSC70 antibody (1:1000 dilution). The overexpression of HSC70 was assessed by immunoblotting with the anti-HSC70 antibody (upper panel). The HIF-1alpha protein levels of mock cells in the absence of NH₄Cl under hypoxic conditions were set to 1.0. (B) PDI/pcDNA 3.1 (+) and si-HSC70 or si-PDI and HSC70/pcDNA 3.1 (+) were transfected into Hep3B cells, cells were cultured for 6 h under hypoxic conditions, and immunoblotting was performed. The HIF-1alpha protein levels of si-ctrl cells were set to 1.0. (C) Hep3B cells overexpressing PDI were cultured for 6 h under hypoxic conditions and then subjected to immunoblotting. Values are expressed as the mean ± S.D. (error bars) of three different plates. N, Normoxia. * p<0.05, ** p<0.01, significantly different from   mock cells (A and C) or si-ctrl cells (B) under hypoxic conditions.  (D) Hep3B cells overexpressing PDI WT or PDI C53, 397S were cultured under hypoxic conditions for 8 h in the presence of NH₄Cl. Cell extracts were subjected to immunoprecipitation (IP) using the anti-HIF-1alpha antibody (Ab). Precipitated proteins were separated by SDS-PAGE, and immunoblotting was performed with the anti-HSC70 or -HIF-1alpha Ab, respectively. Arrows indicate bands corresponding to HSC70 or HIF-1alpha. N, Normoxia; H, Hypoxia; ctrl, control.