Figure 4.

GRP78 mediates ISM internalization and co-targets with ISM to mitochondria. (a) IF showing internalized ISM-GFP co-localized with mitochondria in HUVECs (top panel) and HEK293T cells (bottom panel) transfected with vector coding for ISM-GFP. The medium GRP78-GFP concentration in HUVECs 48 h post transfection is ∼9.7 nM (data not shown). (b) IF showing internalized rISM (his-tagged) co-localized with mitochondria in HUVECs. rISM (1 μM) was added to HUVECs for 24 h before processed for IF. Quantitative measurements of Pearson's correlation coefficient (R_p) between red and green fluorescent labeled channels are indicated in the rightmost panel of each row. (c) ISM from transient transfection was mainly existed in mitochondria of HEK293T cells and HUVECs. GRP78 is co-localized into mitochondria with ISM in ISM-overexpressing HUVECs and HEK293T cells. VDAC was used as the marker for mitochondria; integrin β3 was used as the marker for plasma membrane; PDI was used as the marker for ER; β-actin was used as the marker for cytoplasm. WL, whole-cell lysates; Mito, mitochondrial fraction; PM, plasma membrane fraction; ER, endoplasmic reticulum fraction; C, cytoplasmic fraction. (d) Intracellular rISM resulted from incubation of HUVECs with rISM was mainly detected in mitochondria. GRP78 was co-localized to mitochondria with extracellular rISM in HUVECs. (e) ISM's-binding partners in mitochondrial lysates