Figure 2. TRAMM affects kinetochore... | Rockefeller University Press

Figure 2.

$Figure 2. TRAMM affects kinetochore stability. (A) HeLa cells were fractionated into cytosolic (Cyt) and nuclear (Nucl) fractions and analyzed by Western analysis. A portion of the total lysate (TL) was also included. (B) Chromosomes were purified from HeLa cells that were treated with colcemid. The fraction used for final centrifugation (total lysate) generated supernatant (Spnt) and chromosome (Chr) fractions. (C) Purified mitotic chromosomes were stained for endogenous TRAMM and ACA (+) or with secondary antibodies only (−). Although the majority of chromosomes were not positive for TRAMM staining, some chromosomes showed a TRAMM signal at one or both kinetochores (representative images are shown). Ab, antibody. Bars, 2 µm. (D) HeLa cells were treated with NS or TRAMM-specific siRNA for 24 h. A portion of the cells were fixed and stained with ACA and an antibody against the indicated protein. A second portion was lysed and subjected to Western analysis using an antibody against the indicated protein. Tubulin was used as a loading control. The signal intensity in the table indicates fluorescence intensity of the TRAMM-depleted sample as a percentage of the NS siRNA control ± SD.$

TRAMM affects kinetochore stability. (A) HeLa cells were fractionated into cytosolic (Cyt) and nuclear (Nucl) fractions and analyzed by Western analysis. A portion of the total lysate (TL) was also included. (B) Chromosomes were purified from HeLa cells that were treated with colcemid. The fraction used for final centrifugation (total lysate) generated supernatant (Spnt) and chromosome (Chr) fractions. (C) Purified mitotic chromosomes were stained for endogenous TRAMM and ACA (+) or with secondary antibodies only (−). Although the majority of chromosomes were not positive for TRAMM staining, some chromosomes showed a TRAMM signal at one or both kinetochores (representative images are shown). Ab, antibody. Bars, 2 µm. (D) HeLa cells were treated with NS or TRAMM-specific siRNA for 24 h. A portion of the cells were fixed and stained with ACA and an antibody against the indicated protein. A second portion was lysed and subjected to Western analysis using an antibody against the indicated protein. Tubulin was used as a loading control. The signal intensity in the table indicates fluorescence intensity of the TRAMM-depleted sample as a percentage of the NS siRNA control ± SD.