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. 2014 Oct 1;127(19):4213–4224. doi: 10.1242/jcs.151167

Fig. 6.

Fig. 6.

Mutation of Ser756 to Ala blocks acute LPS-stimulated podosome loss. (A) Itgb2-null BMDCs were infected with retroviral constructs expressing WT-Itgb2-EGFP or S756A-Itgb2-EGFP (green). Cells were treated as indicated with (+LPS) or without (–LPS) 50 ng/ml LPS for 30 minutes, then fixed and stained to visualize F-actin (red; Alexa-Fluor-555) and α-actinin 4 (grey; Alexa-Fluor-633). Podosomes reconstituted with the S756A-Itgb2-EGFP mutant were resistant to the LPS-stimulated disassembly seen for WT-Itgb2-EGFP (A; green). Images were acquired using a Zeiss LSM700, as in Fig. 1. Scale bars: 5 µm. (B) Percentage of EGFP+ cells that contain podosomes when reconstituted with Itgb2-EGFP constructs containing double or single Ser to Ala mutations (S745A and S756A, or either S745A or S756A) with or without treatment with LPS (50 ng/ml) or prostaglandinE2 (10 µg/ml) for 30 minutes, before fixation and staining as above. LPS-driven podosome loss, though significant (**P = 0.005) for WT-Itgb2-EGFP, was not significant in cells expressing the single S745A or S756A β2 mutants (paired t-tests). (C) CD40 expression in cells expressing WT, S745A or S756A Itgb2 was assessed by flow cytometry in control cells (dashed line) and after 20 hours of LPS treatment (solid line).