Figure 4.

Immunoblot of cytoskeletal components in cells continuously kept in random (A, C, E, G, and I), A19 (B, D, F, H, and J), random/2 (K, M, O, and Q), or A19/2 (L, N, P, and R) oligonucleotides. CACO-2 (A–F and K–N) and MCF-10A (G–J and O–R) cells were grown on 24-mm Transwell™ filters and extracted/scrapped from the filter in EB-TX- 100. The pellets were further extracted in SDS sample buffer, and samples from equivalent numbers of cells were run in SDS-PAGE ( the protein in each sample was measured in a small aliquot and the sample volume adjusted to seed 4 μg of protein in all lanes; usually these adjustments represented variations in sample volumes that were <10% of the total volume) and immunoblotted onto nitrocellulose filters. The blots were processed for indirect chemiluminescence using an anti-CK19 mAb (RCK108; A, B, G, and H). The same nitrocellulose sheets used for A, B, G, H, K, L, O, and P were then stripped in SDS/2-mercaptoethanol and reprobed with antiPancytokeratin mAb (AE1/AE3; C, D, I–J, M, N, and O–R). The same filter from CACO-2 extract was stripped and reprobed one more time with anti-actin mAb (C4; E and F). Notice the decrease in CK19 bands in A19 treated cells (B and H) as compared with the control with random oligonucleotide (A and G), and that other cytokeratins and actin in the same samples were not changed (C–F, I, and J). The average OD measures obtained from unfiltered digitized images after subtracting background from the average pixel value over the main band of each lane were as follows (scale 0–255): (A) 82; (B) 12; (C) 77; (D) 100; (E) 91; (F) 81; (G) 30; (H) 6; (I) 28; (J) 21; (K) 150; (L) 27; (M) 171; (N) 172; (O) 49, (P) 5; (Q) 160; (R) 174. The apparent molecular weight of standards is expressed in kD.