Fig. 7

The levels of CDK2m3 mutant protein and vesicles decreased in the MLF knockdown trophozoites. A, C, E, G, I, K Quantification of Cdk2m3 vesicles in the MLF knockdown trophozoites. A pPCdk2m3 expression vector was transfected to the MLF KD and control cell lines (trophozoites stably transfected with pRANneo vector with further removal of G418). The stable transfectants were cultured in growth medium containing (A) 80 μM MG132, (C) 36 μM rapamycin, (E) 100 μM chloroquine, (G) 5 μM nocodazole, (I) 5 mM DTT, and (K) 217 μM G418 for 24 h and then subjected to immunofluorescence analysis using anti-HA antibody for detection. Quantification of CDK2m3-localized vesicles in the treated cells during vegetative stage was performed using Imaris software. **, p < 0.01. ***, p < 0.001 (n = 200–300 cells/condition). ns, p > 0.05, not significant. B, D, F, H, J, L The level of Cdk2m3 protein decreased in the MLF knockdown trophozoites. The MLF KD and control cell lines were used to transfect pPCdk2m3. The stable transfectants were subcultured in growth medium containing (B) 80 μM MG132, (D) 36 μM rapamycin, (F) 100 μM chloroquine, (H) 5 μM nocodazole, (J) 5 mM DTT, and (L) 217 μM G418 for 24 h and then subjected to SDS-PAGE and Western blot analysis. The blot was probed with anti-HA and anti-MLF antibodies, respectively. SDS-PAGE with Coomassie Blue staining is included as a control for equal protein loading. The band intensity from triplicate Western blots was quantified using Image J. The CDK2m3 protein levels were normalized to the loading control (Coomassie Blue-stained proteins). The ratio of CDK2m3 protein levels in drug-treated sample to levels in untreated sample is shown and expressed as mean ± 95% confidence intervals. *, p < 0.05. **, p < 0.01. ***, p < 0.001. ns, p > 0.05, not significant