Fig 2: TfR1 interacts with IKK in cells(A) TfR1 interacts with TAP–IKKβ. TAP-tag-purified material was analysed by Western blotting for the presence of TfR1. (B) HEK-293 cells were transfected with the plasmids indicated and whole-cell lysates were prepared. Anti-GFP antibody-bound beads were used to immunoprecitate GFP–TfR1. Precipitates were resolved by SDS/PAGE and then analysed by Western blotting using the antibodies indicated. (C) Whole-cell lysates were prepared from U2OS cells and immunoprecipitated with normal rabbit IgG or anti-IKK antibodies. Precipitates were resolved by SDS/PAGE and then analysed by Western blotting using the antibodies indicated. (D) U20S cells were treated with 10 ng/ml TNFα for the times indicated before lysis. Whole-cell lysates were immunoprecipitated with normal rabbit IgG or anti-IKK antibodies. Precipitates were processed and analysed as in (C). IP, immunoprecipitation.

Fig 3: Depletion of TfR1 sensitizes cells to TNFα-induced apoptosis(A) U2OS cells were depleted of TfR1, and subsequently stimulated with increasing concentrations (0, 10, 20, 30 and 40 ng/ml) of TNFα for 24 h. Whole-cell lysates were subjected to immunoblotting analysis for the levels of the proteins indicated. NT, non-targeting. (B) U2OS cells were transfected with 1 μg of control or RelA-expression constructs 24 h before TfR1 depletion by siRNA. Cells were treated with 10 ng/ml TNFα for an additional 24 h before lysis. Whole-cell lysates were subjected to immunoblotting analysis for the levels of the proteins indicated.

Fig 4: IL-34 and CSF-1 induce equivalent macrophagic differentiation of primary monocytes. Human peripheral blood monocytes from healthy donors were exposed to 100 ng/mL CSF-1 or 100 ng/mL IL-34 for the indicated times. (a,b) Macrophage differentiation was examined morphologically (fibroblastic shape) and by 2-color flow cytometric analysis. The percentage indicates cells that express both CD71 and CD163. (c) Macrophagic differentiation of monocytes from 3 different healthy donors was followed by 2-color flow cytometric analysis. The percentage indicates cells that express both CD71 and CD163. *P < 0.05 according to a paired student t test.

Fig 5: Depletion of TfR1 impairs IKK complex formation(A) U2OS cells were transiently depleted of TfR1 using siRNA. Whole-cell lysates were subjected to immunoblot analysis for the levels of the proteins indicated. (B) Whole-cell lysates prepared from U2OS cells treated with non-targeting (NT) siRNA or TfR1 siRNAs were immunoprecipitated with normal rabbit IgG or anti-IKKγ or anti-IKKβ antibodies. Precipitates were resolved by SDS/PAGE and then analysed by Western blotting with anti-IKKα, anti-IKKβ and anti-IKKγ antibodies to assess the integrity of the IKK complex. IP, immunoprecipitation.