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. 2001 May 15;107(10):1263–1273. doi: 10.1172/JCI11596

Figure 2.

Figure 2

Homocysteine induces the expression of GRP78/BiP and GADD153 in HepG2 cells. (a) Northern blot analysis of the steady-state mRNA levels of GRP78/BiP and GADD153 in HepG2 cells cultured for 4 hours in the absence (control) or presence of either 5 mM homocysteine, 5 mM cysteine, 5 mM methionine, 10 μg/ml tunicamycin, 2.5 mM DTT, 5 mM homoserine, or 5 mM glycine. Total RNA (10 μg/lane) was size fractionated by agarose-gel electrophoresis, transferred to nylon membranes, and subjected to blot hybridization using radiolabeled cDNA probes encoding human GRP78/BiP or GADD153. Control for equivalent RNA loading was assessed using a radiolabeled GAPDH cDNA probe. (b) Immunoblot analysis of GRP78/BiP and GADD153 protein in HepG2 cells cultured in the absence or presence of 5 mM homocysteine for the indicated time periods. HepG2 cells were also treated with 2.5 mM DTT or 10 μg/ml tunicamycin for 8 hours. Total protein lysates (40 μg/lane) were separated on 12% SDS-polyacrylamide gels under reducing conditions, transferred to nitrocellulose membranes and immunostained with Ab’s against either GRP78/BiP (anti-KDEL) or GADD153.