Fig. 7: Long-term culture of macrophages stimulated with IFN-γ.

a Cells were stimulated with IFN-γ and harvested after 24 h. Two micrograms of each macrophage extract was separated via SDS‒PAGE on a 12% (w/v) acrylamide gel and subjected to Western immunoblotting with antibodies specific for the β5i subunit of the immunoproteasome and the PA28β subunit of the PA28αβ RP. b Native extracts collected after 24 h of IFN-γ stimulation were separated on a 4% (w/v) acrylamide gel under native conditions in the presence of ATP and subjected to western immunoblotting with specific antibodies against the immunoproteasome β5i subunit, PA28β, constitutive β5 subunit and α subunits. sLLVY-AMC and ANW-AMC hydrolysis represent “in-gel peptidase activity”, as determined using fluorogenic substrates for chymotrypsin-like and β5i-associated activities, respectively. After native PAGE, the gels were incubated with the specific substrate as indicated, and the activity was detected in a ChemiDoc system under UV light. c Aggresome (red) formation under basal and IFN-γ-stimulated conditions was detected with a ProteoStat® Aggresome Detection Kit at 7, 14 and 28 days of culture. DRAQ5 was used to visualize the nuclei (blue). Images were acquired with a confocal Leica microscope (×63 oil objective) using a standard rhodamine filter set. Scale bars 20 µm. IB: immune blot.