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

Figure 6.

Figure 6

Overexpression of GRP78/BiP prevents the increase in steady-state mRNA levels of SREBP-1 and IPP isomerase by homocysteine. (a) Immunoblot analysis of GRP78/BiP and GRP94 in wild-type (T24/83), vector-transfected (T24/83-pcDNA), or GRP78/BiP–overexpressing (T24/83-GRP78) cells. Total protein lysates (40 μg/lane) were separated on 10% SDS-polyacrylamide gels under reducing conditions and either stained with Coomassie blue (upper panel) or immunostained with an anti-KDEL mAb. (b) Immunolocalization of GRP78/BiP in the ER of wild-type or GRP78/BiP–overexpressing T24/83 cells. Cells grown on glass coverslips were fixed, permeabilized, and immunostained with an anti-GRP78/BiP polyclonal Ab. ×800. (c) Northern blot analysis of the steady-state mRNA levels of SREBP-1 and IPP isomerase in vector-transfected (T24/83-pcDNA) or GRP78/BiP–overexpressing (T24/83-GRP78) cells cultured in the absence or presence of 1 mM homocysteine for the indicated time periods. 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 SREBP-1 or IPP isomerase. Control for equivalent RNA loading was assessed using a radiolabeled GAPDH cDNA probe.