Figure 5EM micrographs of double immuno-gold labeling using antibodies against immunoproteasome subunit LMP2 and the endolysosomal membrane marker LAMP1 in BMDC, WT MEF and LMP2 KO MEF. Large gold particles (15 nm, black arrow head) labels LMP2 and small gold particles labels LAMP1 (5 nm, white arrow head) (Scale bars = 500nm). The insets are the magnification of region of interest containing LAMP1 positive vacuole (Scale bars = 100nm), marked by the black rectangle.The distribution of LMP2 positive signals (n = 22 images) assessed by plotting the number of gold particles labeling LMP2 within the LAMP1 positive membrane compartment versus those outside the LAMP1 positive organelles.Immuno-gold labeling with antibodies against constitutive proteasome subunits, α5, β5 and 19 S2 (15 nm, black arrow head), co-labeled with an anti-LAMP1 antibody (5 nm, white arrow head) in BMDC (Scale bars = 500nm). Regions of interest containing LAMP1 positive vacuoles are marked by the black rectangles and magnified in the insets (Scale bars = 100nm).Immuno-electron microscopy images (n = 23) were analyzed by plotting the total number of gold particles labeling proteasome subunits inside and outside the LAMP1 positive membrane compartments (D). These data are also presented as the percentage of the total gold particles that are within the LAMP1 positive organelle (E).BMDC transduced with genes encoding GFP, GFP-Rab5ACA and GFP-Rab22ACA under the control of an inducible promoter were fixed and stained for LMP2 and LAMP1 and analyzed by confocal microscopy 24hr after doxycycline induction and 4hr after Alexa 647-OVA coated bead uptake. A single optical section of 10 optical sections of a representative cell are shown (Scale Bar 10μm). Region of interest containing LAMP1 positive vacuole containing beads coated with Alexa-647 conjugated OVA, marked by white rectangles, are magnified as inset (Scale bars = 1μm)BMDC transduced with genes encoding GFP, GFP-Rab5ACA and GFP-Rab22ACA under the control of an inducible promoter were fixed and stained for LMP2 and LAMP1 and analyzed by confocal microscopy 3D-rendering of 10 optical sections of a representative cell are shown (Scale Bar 10μm). Region of interest containing LAMP1 positive vacuole containing beads coated with Alexa-647 conjugated OVA, marked by white rectangles, are magnified as inset (Scale bars = 1μm)The same cells were analyzed for LMP2 fluorescence intensity within the phagosomal lumen as defined by a region of interest positive for Alexa 647-OVA enclosed within a limiting membrane positive for LAMP1. The fluorescence intensity of LMP2 within the phagosomal lumen normalized to Alexa647-OVA was calculated and data compiled from independent experiments (n=3) are plotted (H).For each transduced cell type, images of at the least 27 cells acquired across three independent experiments were analyzed and each cell is represented as a data point.BMDC co-expressing LMP2-RFP and GFP-Rab5ACA were live imaged 16hr after doxycycline induction and 30min post uptake of Alexa647 coated latex beads (Bar 10μm). Images were acquired every 3min and images taken at 12min interval are presented (I-K). The arrowheads in panel (I and J) mark the phagosome or vacuoles containing LMP2, respectively.BMDC co-expressing LMP2-RFP and GFP-Rab22ACA (J, K) were live imaged 16hr after doxycycline induction and 30min post uptake of Alexa647 coated latex beads (Bar 10μm). Images were acquired every 3min and images taken at 12min interval are presented (I-K). The arrowheads in panel (I and J) mark the phagosome or vacuoles containing LMP2, respectively. The boxed region in panel (J) marks a phagosome that has been enlarged in panel (K) (Bar 1μm).