Journal: The Journal of Cell Biology

Article Title: In Vitro Reconstitution of Cortical Actin Assembly Sites in Budding Yeast

doi:

Figure Lengend Snippet: Sequential activation of actin assembly sites by ACF1 and ACF2. ( A ) An extract was fractionated over a Q-Sepharose column to yield the flow through and the 0.5 M KCl eluate, which were subsequently desalted and concentrated. The urea-treated, permeabilized cells were incubated with the flow through ( a ), the eluate ( b ), or a 1:1 mixture of the two fractions ( c ) before Rd-actin polymerization. Rhodamine fluorescence images of representative groups of cells are shown. ( B ) The complementing factors in the flow through and in the eluate are designated ACF1 and ACF2, respectively. The histograms show the percentages of the urea-treated cells that preferentially incorporated Rd-actin into the bud after the cells were incubated with the buffer, a mixture of ACF1 and ACF2, ACF1 first and then ACF2, or ACF2 first and then ACF1. In the latter two treatments, the cells were washed with the buffer after the incubation with the first factor. The percentages shown are averages of the results from two experiments, and the error bars are standard deviations. Bar, 10 μm.

Article Snippet: 1-liter extract (15 mg/ml) from 0.4 kg of wet RLY1 cell pellet was loaded onto a 400-ml Q Sepharose column (Macro-Prep Q; BioRad Laboratories) equilibrated with UB plus PI.

Techniques: Activation Assay, Incubation, Fluorescence

Journal: The Journal of Cell Biology

Article Title: In Vitro Reconstitution of Cortical Actin Assembly Sites in Budding Yeast

doi:

Figure Lengend Snippet: Bee1 is an essential component of ACF1. An extract prepared from the wild-type (RLY1) or the Bee1-myc–expressing strain (RLY160) was passed over a Q-Sepharose column yielding the flow through ( FT ). The bound proteins were eluted with 0.25 M KCl to yield P1 and then with 0.4 M KCl to yield P2. ( A ) Immunoblot analysis with a mouse anti-myc antibody showing that Bee1-myc is mostly in P2 ( a ) and showing the depletion of Bee1-myc from P2 by the anti-myc affinity beads but not by the control anti-HA affinity beads ( b ). ( B ) The histograms show the percentages of the urea-treated cells that preferentially incorporated Rd-actin into the bud after the cells were incubated under the conditions described in the table below. The cells were washed with the buffer between the two incubation steps. The percentages shown are averages of the results from two experiments, and the error bars are standard deviations. wt-ext , An extract from the wild-type strain (RLY1); Δb1-ext , an extract from the Δbee1 strain (RLY157); Bm , the Bee1-myc–expressing strain (RLY160); HA-dpl , after depletion with the control anti-HA affinity beads; myc-dpl , after depletion with the anti-myc affinity beads. ( C ) The order of interaction between Bee1 and the unknown ACF1 component. The histograms show the percentages of the urea-treated cells that preferentially incorporated Rd-actin into the bud after the cells were incubated under the conditions described in the table below. The cells were washed with the buffer between the two incubation steps. The percentages shown are averages of the results from four experiments, and the error bars are standard deviations. The abbreviations are the same as in B.

Article Snippet: 1-liter extract (15 mg/ml) from 0.4 kg of wet RLY1 cell pellet was loaded onto a 400-ml Q Sepharose column (Macro-Prep Q; BioRad Laboratories) equilibrated with UB plus PI.

Techniques: Expressing, Western Blot, Control, Incubation

Journal: The Journal of Cell Biology

Article Title: In Vitro Reconstitution of Cortical Actin Assembly Sites in Budding Yeast

doi:

Figure Lengend Snippet: Purification of ACF2. The peak fractions that contained ACF2 activity after each consecutive purification step (from left to right) were analyzed on 12.5% SDS–polyacrylamide gels. The gels were stained with Coomassie blue and photographed. M , protein molecular weight marker (Life Technologies, Inc., Gaithersburg, MD). E , the starting extract; Q , Q-Sepharose column; AS , 45% ammonium sulfate precipitation; H , heat treatment at 60°C; S , S-Sepharose and heparin columns (connected in series); HI , methyl hydrophobic interaction column; GF , S-200 Gel filtration column; Q2 , Bio-Scale Q2 column.

Article Snippet: 1-liter extract (15 mg/ml) from 0.4 kg of wet RLY1 cell pellet was loaded onto a 400-ml Q Sepharose column (Macro-Prep Q; BioRad Laboratories) equilibrated with UB plus PI.

Techniques: Purification, Activity Assay, Staining, Molecular Weight, Marker, Filtration