Dynamin inhibition affects secretion of other proteins.

HMDM were exposed to 30 µM MiTMAB for up to 2 h. Total protein secreted (A) was determined by TCA-precipitation of [35S]-labelled proteins from medium of experiments described in Fig. 4. (B) Ctrl and TKO mouse fibroblasts were generated by treatment without and with 4-hydroxytamoxifen for 7 days, respectively. Cellular Dynamin II levels were determined by Western Blotting and corrected for α-tubulin to account for uneven loading of cell material. (C) Ctrl and TKO mouse fibroblasts were incubated in methionine/cysteine free DMEM with 250 µCi/mL [35S]methionine/cysteine for 2 h. Cells were then exposed to 30 µM MiTMAB, 30 µM Dyngo-4a and 10 µM Dynole-34-2 after which total TCA-precipitable counts were determined in medium and cell lysates. Counts are corrected for protein amounts and expressed as a percentage of the initial [35S]-precipitable material at T = 0. (D) Phosphorimager image showing [35S]- proteins secreted from Ctrl and TKO cells. Lanes were corrected for protein amounts in respective wells and specific activity. Molecular weight markers are annotated. Arrows indicate decreased protein secretion from TKO, asterisks represent proteins with similar protein secretion between Ctrl and TKO cells. Data shown are mean ± SD from triplicate cultures representative of at least 2 experiments. *Pv Ctrl.