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. 2001 Jul 10;98(16):9128–9132. doi: 10.1073/pnas.161283998

Figure 2.

Figure 2

Immunodepletion of Plk from mitotic cytosol inhibits Golgi fragmentation. (A) Semi-intact, salt-washed NRK cells were incubated in the presence of an ATP-regenerating system with either 7 mg/ml interphase cytosol (IE), 7 mg/ml mitotic extract (ME), or 7 mg/ml mitotic extract depleted of Plk (ME-depleted). After a 60-min incubation at 32°C, cells were fixed and stained with anti-mannosidase II antibody to visualize the Golgi apparatus by fluorescence microscopy. (B) Immunoblot of mock (m) and Plk-depleted (d) mitotic extracts. Greater than 90% depletion of Plk was achieved by two consecutive rounds of incubation of mitotic extract with anti-Plk antibody conjugated to protein G-Sepharose beads. (C) Percentage of cells with fragmented Golgi after incubation of semi-intact cells with either mock (ME mock) or Plk-depleted (ME-depleted) mitotic cytosol or Plk-depleted cytosol to which 20 μg/ml constitutively active recombinant Plk (ME-depleted + Plk-active) was added. The data represent the average of four independent experiments.