Figure 7

DEPP is located in mitochondria and peroxisomes. a) SH-EP/tetEGFP and SH-EP/tetEYFP-DEPP cells were treated with 200 ng/ml doxy for 24 hours and subjected to subcellular fractionation. The protein levels of DEPP, GFP and catalase in the cytosolic and the nuclear fraction were determined by immunoblot. Alpha-Tubulin (cytosolic) and Lamin (nuclear) were used to control the purity of the fractions (left panel). The peroxisomal fraction of SH-EP/tetEYFP-DEPP cells treated with 200 ng/ml doxy for 24 hours was separated using a peroxisome isolation kit (PEROX1, Sigma). DEPP protein expression was determined by immunoblot. Catalase (peroxisomes) and CoxIV (mitochondria) were used to control the purity of the peroxisomal fraction (right panel). b) SH-EP/tetEYFP-DEPP cells were grown on eight-well chambered cover glasses, treated with 200 ng/ml doxy for 24 hours and analyzed by live confocal microscopy. Mitochondria were stained using the specific mitochondrial probe MitoTrackerRed/CMXRos (30 nM). Peroxisomal staining was performed with a CellLight® Peroxisomes-RFP fusion construct. c) SH-EP/tetDEPP cells treated with 200 ng/ml doxy for 24 hours were subjected to CoIP analysis with a Pierce Crosslink Magnetic IP/Co-IP kit to investigate whether the DEPP protein co-immunopurifies with PEX7-cross-linked beads. The expression of PEX7 and DEPP was determined by immunoblot. Alpha-Tubulin served as loading control.