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. Author manuscript; available in PMC: 2013 Oct 3.
Published in final edited form as: Nat Commun. 2013;4:1659. doi: 10.1038/ncomms2669

Figure 1. Depletion of GRASP55/65 does not cause apoptosis or ER stress.

Figure 1

(A) HeLa cells transfected with the indicated RNAi were stained with annexin V-GFP before fixation. Note that no obvious annexin V staining was observed on GRASP-depleted cells. (B) HeLa cells treated with 2 µM staurosporine (Stau) for 0 h or 2 h were stained with annexin V-GFP before fixation. Scale bars in A-B, 10 µm. (C) HeLa cells transfected with the indicated RNAi or treated with staurosporine (ST) were analyzed by Western blot. Note that the cleaved caspase 3 was detected after 2 h of staurosporine treatment, but not following the depletion of the GRASPs. (D) Depletion of GRASPs does not cause ER stress. HeLa cells transfected with the indicated RNAi or treated with DMSO or 2 µM thapsigargin (Thapsig) for 4 h or 16 h were analyzed by SDS-PAGE and Western blot. Note that the protein level of Bip increased after thapsigargin treatment, but not following GRASP depletion. (E) GRASP depletion does not result in ERAD. HeLa cells transfected with indicated RNAi were first treated with 5 µg/ml BFA for 1 h followed by the addition of 100 µM cycloheximide (CHX) into the medium and incubated for the indicated time period. Shown are Western blots of cell lysate for β1 integrin and p97 on the same gels. Molecular weight standards (kDa) are indicated on the left in C–E.