Fig 2: Minimal requirements for the investigation of Aβ interactions with FPRs. Aβ, amyloid beta; FPR, Formyl peptide receptor.

Fig 3: DMSO influences FPR activation by Aβ1-42. Mean Ca2+ peak responses of human (red) or mouse (blue) FPRs to 5 μM of Aβ1-42 peptides obtained from P&E, Synpeptide or Anaspec dissolved in either DMSO (striped bars) or C1 (clear bars), n=3, N=2. All Error bars, S.D.; ∗, p ≤ 0.05; ns, no significance.

Fig 4: Validation of immunofluorescence staining with FPR subtype-specific antibodies. Top: Comparison of immunofluorescence staining intensity of FPR-transfected HEK293T and U87 cells with receptor subtype specific antibodies. The hFPR1 (1 μg/ml), hFPR2 (0.2 μg/ml) and hFPR3 (1 μg/ml) antibodies were used. Note that the absolute staining intensities of all hFPRs in HEK293T cells were highly similar, however a strong reduction of hFPR2 staining in U87 cells is observed. No cross reactivity was detected. All images were acquired with the same settings and are displayed with the same illumination, brightness and contrast. Evidence for similar cell surface expression and production rates for hFPR1 and hFPR2 can be found in supporting Fig. S8. Bottom: corresponding nuclei staining with Hoechst 33342 demonstrating a comparable amount of total cell number in all visual fields.

Fig 5: Evidence for similar cell surface expression and calcium responses of FPR1 and FPR2 in HEK293T cells. A, HEK293T cells were transfected with varying amounts of plasmid DNA for hFPR1 or hFPR2 and subsequently treated with 1 μM of FITC-labeled WKWVm-NH2 to monitor their cell surface expression. Left: representative images of treated cells transfected with different DNA amounts Right: quantitative analysis of positively stained cells. Note that cell surface expression of both receptors upon dilution is reduced in a similar manure. Error bars, S.D. n=1, N=3 B, Mean Ca2+ peak responses of HEK293T cells transfected with decreasing amounts of human (red) or mouse (blue) FPR plasmid DNA. FPR1 and 2 of each species were treated with 1 μM of WKWVm-NH2 and FPR3-transfected cells with 30 μM of WKWVm-CHO. Note that the amount of FPR 1 and 2 DNA can be diminished by more than 10fold without a strong reduction of the signal amplitudes.