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MATERIALS AND METHODS

By Ingrid Blikstad, Lars Carlsson and Cell Culture

Abstract

ABSTRACT We measured the pools of unpolymerized and filamentous actin in homogenates of HeLa cells made in several different lysis buffers, as well as after treatment of cells with a variety of chemicals or trypsin, and after adenovirus (type 2) infection. This was possible when a series of factors concerning the basic culture conditions were kept constant: e.g., serum type used, serum batch, cell density, time after subcultivation of cells, and buffering substance in the medium. Homogenates from untreated cells usually contain 35-45 % of the total actin in an unpolymerized form. With some batches of cells this number can be as high as 50 %. In sparse cultures (3 x 10 ° cells/cm'), HeLa cells contain ^-10 pg actin/cell, while the corresponding number is only 5 pg in dense cultures (3 x 105 cells/Cm 2). Treatment of cells with cytochalasin B increases the pool of unpolymerized actin by-30-40%, while colchicine decreases the fraction of unpolymerized actin by 20 %. The oxidant diamide increases the filamentous actin pool 25-50 %. Glucose, sodium azide, dinitrophenol, serum starvation, or thymidine treatment does not affect the distribution between unpolymerized and filamentous actin to any significant extent. Trypsin and EDTA induced rounding up of cells but did not change the actin distribution. The distribution of actin between G- and F-forms was unchanged after adenoviru

Year: 2013
OAI identifier: oai:CiteSeerX.psu:10.1.1.318.1940
Provided by: CiteSeerX
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