Journal: The Journal of Biological Chemistry

Article Title: ERp57 Modulates STAT3 Signaling from the Lumen of the Endoplasmic Reticulum *

doi: 10.1074/jbc.M109.054015

Figure Lengend Snippet: Western blot and microscopy analysis of ERp57-deficient cells. A, Western blot analysis of wild-type, ERp57-deficient cells (ERp57−/−), and ERp57−/− cells expressing full-length recombinant ERp57 (ERp57−/−-ERp57ER) or cytoplasmic-targeted, no signal sequence ERp57 (ERp57−/−-ERp57cyt). Blots were probed with rabbit anti-ERp57 antibodies (lower protein band corresponds to a nonspecific immunoreactivity) and anti-GAPDH antibodies. In B, immunostaining of wild-type cells (wt), ERp57-deficient cells (ERp57−/−), ERp57-deficient cells expressing full-length, ER-targeted recombinant ERp57 (ERp57−/−-ERp57ER), ERp57-deficient cells expressing cytoplasmically targeted recombinant ERp57 (ERp57−/−-ERp57cyt), calreticulin-deficient cells (crt−/−), calreticulin-deficient cells expressing full-length calreticulin (crt−/−-CRT), calreticulin-deficient cells expressing loss of ERp57 binding mutant (crt−/−-CRTE239R) or ERp57 binding mutant of calreticulin (crt−/−-CRTG242A). Cells were stained with anti-ERp57 antibodies and Texas Red-conjugated concanavalin A. In each panel images represent staining with anti-ERp57 antibodies, ConA staining, phase-contrast, and merged images of the cells. Scale bar = 16 μm. C, Western blot analysis of cell lysate, cytoplasmic, and microsomal (containing ER) fractions of wild-type, ERp57-deficient (ERp57−/−), and ERp57-deficient cell lines expressing cytoplasmic targeted ERp57 (ERp57−/−-ERp57cyt). D, flow cytometry analysis of specific cell lines was carried out with anti-ERp57 antibodies. Wild-type, ERp57−/−-Erp57ER, ERp57−/−-Erp57CYS, crt−/−, crt−/−-CRT, crt−/−-CRTE239R, crt−/−-CRTG242A cell lines were used for the analysis. Results are presented as the relative mean florescence intensity after subtracting unspecific staining of ERp57−/− cells. M2 represents the gate set on cells stained with antibody. E, electron microscopy analysis of wild-type and ERp57-deficient cells. The arrows indicate the location of the ER. Scale bar = 0.2 μm. F, Western blot analysis of ER chaperone proteins and oxidoreductases in wild-type (wt) and ERp57-deficient (ERp57−/−) mouse embryonic fibroblasts. Antibodies used to probe the Western blot are indicated to the right of each panel. Lower protein band in the ERp57 lanes corresponds to a nonspecific immunoreactivity. CNX, calnexin; CRT, calreticulin.

Article Snippet: The coverslips were mounted onto glass slides and fluorescent signals visualized using spinning disk confocal microscopy (WaveFX from Quorum Technologies, Guelph, Canada) set up on an Olympus IX-81 inverted stand (Olympus, Markham, Canada).

Techniques: Western Blot, Microscopy, Expressing, Recombinant, Sequencing, Immunostaining, Binding Assay, Mutagenesis, Staining, Flow Cytometry, Electron Microscopy