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

Fig. 6.

Fig. 6.

Total hepatic protein ubiquitination and effects of acute hepatic heme depletion and repletion on hepatic content of ER-chaperones Grp78 and Grp94 in cultured MGB wild-type [WT; HRI (+/+)] and HRI knockout [KO; HRI (−/−)] mouse hepatocytes. A, untreated WT or KO mouse hepatocytes pooled from two mice each were cultured as detailed under Materials and Methods for each individual experiment. Hepatocyte lysates (50 μg of protein) from four individual experiments were prepared, and total hepatic protein ubiquitination was examined by Western immunoblotting analyses as described under Materials and Methods. B, WT or KO mouse hepatocyte cultures were untreated, treated with heme (20 μM; H-control), heme (H)-depleted, or heme-repleted after heme depletion, as in Fig. 5C. Aliquots (25 μg of protein) of these same SDS-PAGE sample buffer-solubilized cell lysates were used for Grp78 immunoblotting analyses and other aliquots (10 μg of protein) of these same SDS-PAGE sample buffer-solubilized cell lysates used for actin immunoblotting analyses as loading controls (Top). The densitometric quantification of the relative immunochemically detectable Grp78 content in KO (solid bars) to the WT (open bars) is shown below. Values represent mean ± S.D. of three separate experiments. Statistically significant differences in Grp78 content between the two mean ± S.D. values each marked with the same symbol were as follows: *, p < 0.05; §, p < 0.001; and ‡, p < 0.001. C, aliquots (25 μg of protein) of these same SDS-PAGE sample buffer-solubilized cell lysates were used for Grp94 immunoblotting analyses and other aliquots (10 μg of protein) used for actin immunoblotting analyses as loading controls (top). The densitometric quantification of the relative immunochemically detectable Grp94 content in KO (solid bars) to the WT (open bars) is shown below. Values represent mean ± S.D. of three separate experiments. Statistically significant differences in Grp94 content between the values were as indicated. The difference in basal Grp94 content of untreated WT and KO hepatocytes (*) was statistically significant at p < 0.001.