Fig. 2.

Expression and intracellular processing of the constructs. (A) Built confocal images showing one clone expressing each construct. (i) Green fluorescence shows that the control GFP is localized diffusely throughout the cells, whereas SPARC–GFP and the deletion mutants are localized perinuclearly. (ii) TGN-46 immunostaining indicates the trans-Golgi Network. (iii) Merged images indicate co-localization of TGN-46 with SPARC–GFP and both deletion mutants, confirming their localization to the Golgi complex. (iv) Zoomed images better demonstrate co-localization. Images in i–iii were captured at ×60. Zoomed images are ×240. (B) Western blot analyses of cell lysates (i and iii) and conditioned medium (ii and iv) demonstrate the level of intracellular and secreted constructs and endogenous wt-SPARC by the clones. The parental cell line, U87MG, is represented in lane 1 of each blot. (i and ii) Anti-SPARC antibody detects endogenous SPARC, SPARC–GFP and ΔEGF but not ΔAcidic or GFP. The intermediate bands are believed to be due to alternate processing or proteolytic cleavage. (iii and iv) Anti-GFP antibody detects all of the constructs (GFP is not secreted). Actin indicates equal loading of cell lysates. Molecular weights (in kDa) are shown at the left of each blot.