Fig. 4

ATG5 and UB western blot analyses with protein extracts from L. m.-infected and HBSS-starved BMDM as well as determination of the infection rates of L. m.-infected BMDM after ATG5 and UB downregulation by RNA interference. Methods: (a, b) BMDM from BALB/c mice were infected with L. m. promastigotes for 1 and 24 h. Uninfected control BMDM were incubated for the same time in RPMI medium or starved for 1 h in HBSS. The proteins were harvested and subjected to western blot analysis with specific antibodies against ATG5 and UB. Western blots with proteins from 3 independent experiments were analyzed densitometrically. ACTB served as the internal loading control. c Additionally, BMDM were transfected with specific siRNAs 4 h prior to infection to downregulate the expression of ATG5 and UB. The cells were finally infected with L. m. promastigotes. L. m.-infected control BMDM were transfected with negative control siRNA. The infection rates were determined 48 h p.i.. Diagram shows the result of 2 independent experiments. Results: (a) ATG5 and UB levels in samples from L. m.-infected BMDM 24 h p.i. were increased compared to uninfected control BMDM. b Results of the densitometric analyses confirmed that ATG5 and UB were significantly increased in L. m.-infected BMDM 24 h p.i. compared to uninfected control BMDM. No upregulation was detected in L. m.-infected BMDM 1 h p.i. compared to the respective controls. c A significant increase in the infection rate was found in L. m.-infected BMDM after downregulation of the protein expression of ATG5 or UB compared to L. m.-infected BMDM transfected with negative control siRNA. L. m.-inf. = L. m.-infected, neg. control = negative control, n.s. = not significant, * p ≤ 0.05, *** p ≤ 0.001