Figure 6.

ADF and cofilin1 knockdown causes missorting of soluble proteins. A schematic presentation of the SILAC experiment. HeLa cells were incubated in medium containing normal arginine and lysine or with the stable isotope arginine (13C6) and lysine (13C6/15N2). After five passages, heavy-labeled cells were transfected with control siRNA and unlabeled cells with ADF + cofilin1 siRNA. 48 h after transfection, cells were washed and incubated in medium without FCS and cultured for 2 h. Media were collected and mixed in 1:1 ratio according to cell number and SILAC-based analysis of the cell pellet. The mixed proteins were hereafter separated by 1D gel electrophoresis, and excised bands were trypsinized. Extracted peptides were analyzed by reverse-phase HPLC coupled to a mass spectrometer. Dotted lines together with arrow lines are used to indicate mass differences between SILAC peptide pairs. Based on the relative abundance ratio of specific peptides (Ong and Mann, 2006; Harsha et al., 2008), the proteins were classified as class A (ratio between heavy [H] and light [L] peptides = 1; secretion unchanged), class B (ratio between H/L > 1; secretion inhibited in ADF/cofilin-depleted cells), and class C (ratio between H/L < 1; secretion accelerated in ADF/cofilin-depleted cells).