Methods for Monitoring Endoplasmic Reticulum Stress and the Unfolded Protein Response
Figure 2
Detection of protein levels of UPR target genes. (a) Immunohistochemical detection of CHOP, GRP78, and XBP1 in Multiple Sclerosis patient postmortem brain tissue. Representative images showing upregulation of CHOP (ii) at the edge (LE) of a chronic active lesion, in comparison to (i) NAWM. GRP78 expression was downregulated in the center of a chronic active lesion (iii) when compared to the edge (iv) of actively demyelinating lesions. Sample images illustrate the variety of morphologically distinct cell types that express CHOP or GRP78 including macrophages (M), astrocytes (a), and oligodendrocytes (o). Increased expression of XBP1 was found at the edge of a chronic active lesion (vi), when compared to normal-appearing white matter (v). XBP1 immunostaining is also apparent in a large number of oligodendrocytes (o). All immunoperoxidase-stained cells were detected using the chromogen, DAB (brown), and counterstained with hematoxylin for nuclei (blue). Scale bars = 250 m. Letter codes are as follows: NAWM = normal-appearing white matter; LC = lesion center; LE = lesion edge. Red astrices indicate location of lesion within brain sections analyzed. (b) PC12 cells were treated with 0.25 M of Tg for 0, 2, 4, 6, and 8 hours. Whole cell lysates were analyzed by Western Blot for GRP78, CHOP, spliced XBP1, phospho-eIF-2, and total- eIF-2. -Actin was used to determine equal loading of samples.
