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. 2010 Apr;77(4):575–592. doi: 10.1124/mol.109.061259

Fig. 8.

Fig. 8.

Effects of acute hepatic heme depletion and repletion on hepatic HRI content and autophosphorylation, and eIF2α kinase activity in cultured MGB wild-type [WT; HRI (+/+)] mouse hepatocytes with and without PB pretreatment. A, WT mouse hepatocyte cultures were untreated, treated with heme (H-control), heme-depleted (H-depleted), or heme-repleted after heme depletion (H-repleted) (first four lanes), or pretreated with PB (next three lanes) alone (lane 5), heme-depleted (H-depleted; lane 6), or heme-repleted after heme depletion (H-repleted ; lane 7), as detailed under Materials and Methods. A representative example of HRI and HRI-P Western immunoblotting analyses of these hepatocyte lysates (100 μg of protein) is shown at the top, with corresponding smaller aliquots (10 μg of protein) of these same SDS-PAGE sample buffer-solubilized cell lysates used for actin immunoblotting analyses as loading controls. Densitometric quantification of total hepatic HRI and HRI-P content from three individual experiments is shown at the bottom. Statistical analyses revealed significant differences in both hepatic HRI and HRI-P content between untreated and PB-pretreated at p < 0.001, and between PB/H-repleted and PB/H-depleted at p < 0.05. No statistically significant differences were observed between PB/H-depleted and PB-pretreated or between PB-pretreated and PB/H-repleted WT lysates. B, WT or KO mouse hepatocyte cultures were untreated or pretreated with PB alone (lane 2) and then either heme-depleted (lane 3) or heme-repleted after heme depletion (lane 4), as detailed under Materials and Methods. A representative example of total eIF2α and eIF2αP Western immunoblotting analyses of these hepatocyte lysates (10 μg of protein) is shown at the top with corresponding aliquots used for actin immunoblotting analyses as loading controls. Densitometric quantification of total hepatic eIF2α and eIF2αP content, and corresponding statistically significant differences between mean ± S.D. of 3 individual experiments are shown at the bottom. Statistically significant differences in eIF2α or eIF2αP content were observed between the two mean ± S.D. values each marked with the same symbol as follows: *, p < 0.001; §, p < 0.001; ‡, p < 0.001; =, p < 0.001; ●, p < 0.001; and **, p < 0.001.