Figure 5.

Immunofluorescence images of dual-labeled cells reveal the coexistence of endogenous AtPex16p in peroxisomes and ER. A to O, Representative confocal optical sections of nontransformed Arabidopsis cells processed for microscopy via the on-slide procedure whereby formaldehyde-fixed and pectolyase-cellulase-digested cells were spread on microscope slides, membranes were permeabilized with Triton X-100, and then primary and secondary antibodies and/or Concanavalin A-Alexa 594 were added to the cells (rather than to fixed/permeabilized cells in microfuge tubes as was done for cells shown in Fig. 4). A to C: A, Green Cy2 punctate (arrows) and reticular patterns (arrowheads; rabbit anti-AtPex16p-42 IgGs; 1:10, overnight). B, The same cells exhibit a Cy5 punctate peroxisomal pattern (mouse anti-catalase monoclonal IgG; 1:500, 1 h). C, Merged image shows colocalized (yellow) catalase and AtPex16p in peroxisomes. D to F: D, Different portion of the same population of cells revealed similar Cy2 punctate (arrows) and reticular patterns (arrowheads; rabbit PA anti-AtPex16p IgGs; 1:500, 1 h). E, Same cells with Cy5 peroxisomes (mouse anti-catalase IgGs; 1:500, 1 h). F, Merged image shows colocalized catalase and Atpex16p in peroxisomes. G to I: G, Different portion of the same population of cells (rabbit PA anti-AtPex16p IgGs; 1:500, 1 h). H, Same cells (mouse anti-BiP monoclonal antibodies; 1:500, 1 h). I, Merged image produced a yellow reticulate pattern indicative of ER localization of AtPex16p. J to L: J, Reticulate pattern (PA anti-AtPex16p IgGs; 1:500, 1 h). K, Reticulate pattern (Concanavalin A-Alexa 594; 1:500, 1 h). L, Merged image (yellow) indicative of the ER localization of AtPex16p. M to O. M, Reticulate pattern (mouse anti-BiP monoclonal antibodies; 1:500, 1 h). N, Reticulate pattern (Concanavalin A-Alexa 594; 1:500, 1 h). O, Merged image produced a yellow reticulate pattern demonstrating that both Concanavalin A and BiP are colocalized in the same ER compartment. Bar = 5 μm.