Fig. 2. Endogenous MIR1 is localized at the centrosome. (A) To test the suitability of MIR1 antibodies in immunofluorescence experiments, GFP-MIR1 was expressed in BHK cells. Methanol-fixed cells were processed for immunofluorescence with either Ab284 (left column) or Ab284 that had previously been preadsorbed to recombinant MIR1 protein (right column). The arrow points to a cell that shows GFP fluorescence but no staining with the pre-saturated antibody. Exposure times and image processing parameters were kept constant for all four panels to allow direct comparison (63xfield). (B) Endogenous MIR1 in U2OS cells was visualized after fixation with methanol by immunofluorescence microscopy using Ab284. A low magnification view (upper right panel) shows prominent staining in the perinuclear region. A control shows staining with Ab284 that had previously been preadsorbed to recombinant MIR1 protein (lower right panel) (40x field). Bar 10 µm. A higher magnification picture is shown to the left. Co-staining with an antibody to acetylated tubulin reveals the location of the centrosome (arrows) (100x field). (C) MIR1 staining in different cell types was analyzed after fixation in methanol by immunofluorescence with Ab284 (rhodamine-conjugated secondary antibody). The cells were also stained with mAb GTU-88 to -tubulin (AlexaFluor-488-conjugated secondary antibody). Images, which were acquired at 100x on a Deltavision system, present a collapsed view of multiple processed sections. Bar, 5 µm. (D) MIR1 localization in U2OS cells persists at the centrosome in the absence of a microtubule network. MIR1 was stained with Ab284 and -tubulin with mAb GTU-88. (E) MIR1 localization with Ab520. Fixed U373 cells were stained for MIR1 with Ab520 and for centrosomes with an -centrin antibody (left panels). Samples stained with M1 and Ab520 reveal MIR1 localized along individual microtubules as distinct spots (arrows in right panels, enlarged view). Images, acquired at 100x on a Deltavision system, are of a particular Z-section and have been processed by with an iterative deconvolution algorithm. (F) MIR1 localization in U373 cells during different stages of the cell cycle. Fixed cells were stained for MIR1 with Ab284 anf for centrosomal antigens with human autoimmune serum 5051. The cells were also stained for DNA with Hoechst dye 33258. The arrow shows a cell going through the cell cycle. The arrowhead in the last panel shows another cell loss of MIR1 localization at the spindle poles in metaphase (100x field). (G) A higher magnification view of U373 cells in telophase. Staining was performed with Ab284 for MIR1 and with mAb GTU-88 for -tubulin. DNA was stained with Hoechst dye 33258. A Deltavision image was analyzed as in Fig. 1C. MIR1 staining is present at the centrosomes (arrows) and at the midbody (arrowhead) (100x field).