Matrix metalloproteases MMP 1 and MMP 13 are downregulated in CKII site mutant C4-1 cell lines.

(A) Confluent wild type and CKII site mutant C4-1 cell lines were incubated in a serum-free medium for 48 hours. The conditioned media from the cell lines were concentrated and equal amounts of protein run on non-reducing 10% SDS-PAGE containing 0.1% gelatin, The gel was then renatured and incubated overnight at 37°C for gelatin hydrolysis. After incubation, the gel was stained with Coomassie Blue to visualize the bands of hydrolyzed substrate. (B) Equal amounts of concentrated conditioned media from the indicated cell lines were analysed by western blotting for MMP13 (upper panel). The lower panel shows the Ponceau stain of the nitrocellulose to confirm equal protein loading. Panels (C, D, E, F, G) Cells were processed as in panel B, but with the addition of 2.5 μM of CX-4945, as indicated, to specifically inhibit CKII activity. Conditioned media from wildtype C4-1 and mutant cells, plus CaSki and C33a cells, were then processed as before and analysed by western blotting for MMP13, MMP1 and MMP8 (upper panels), with the lower panels in each case showing the Ponceau stain of the nitrocellulose membranes.