Fig. 3

Release of CCL4/MIP-1β by stimulated NK cells. NK cells were prepared and stimulated as described in methods. a Supernatants were incubated with αCCL4/MIP-1β and A/G sepharose beads, culture medium was included as negative control (−). Immunoprecipitated proteins were analyzed by SDS page and subsequent Western Blot for CCL4/MIP-1β protein. 100 ng (#) and 100 pg (+) rhCCL4/MIP-1β were provided as positive controls. Two lanes were used for molecular weight standards (PR), results are indicated in kDa on the very left. Another two lanes were not used (w/o). For your convenience, the whole X-ray film is shown, the size of the membrane is marked with “|--”. Please note the exposition time on the right corner (1 min) and the appearance of high molecular weight aggregates within the overloaded positive control (#). b The experiment described in (a) was performed four times in total and analyzed by ImageJ. Means and standard deviations were summarized into one diagram. c Cytokine release was blocked by Brefeldin A. CCL4/MIP-1β expression was analyzed by intracellular FACS analysis (colored, thick lines) including isotype controls (thin, black lines) as shown here in a representative result. d The experiment described in (c) was performed three times in total. MFI values were corrected as described in the method section, means and standard deviations were summarized into one diagram. e Supernatants of stimulated NK cells were analyzed by ELISA for CCL4/MIP-1β. The number of samples is indicated for each stimulation condition. Individual results as well as means and standard deviations are depicted. *p < 0.05