Figure 5. sCD89 interacts with TfR1 and induces up-regulation of its expression on mesangial cells. (A–C) Frozen kidney sections immunostained for mouse TfR1 from 12-wk-old WT, α1KI, and α1KI-CD89Tg mice (A), 6-wk-old α1KI mice injected i.v. with BSA or sCD89 (B), and NOD.SCID mice injected with BSA or sCD89 (C). Arrows indicate TfR1+ cells. The corresponding graphs represent the numbers of positive cells per glomerulus counted in 20 randomly chosen fields for each mouse at 200 magnification. n = 3 mice per group. Bars: (A) 10 µm; (B and C) 5 µm. (D) Flow cytometry histogram (one representative experiment of six) of HMCs binding sCD89 (white layer) and stained with anti-CD89. The dashed layer represents incubation with sTfR1. The gray layer corresponds to the isotypic-matched control. The inset graph shows the mean binding values of indicated doses of sCD89 (±SEM) to HMCs as a ratio to background mean fluorescence intensity of seven independent experiments. (E) Flow cytometry analysis of sCD89 binding on HMCs untreated (white layer) or treated with TfR1 miRNA (dotted layer). The inset shows flow cytometry analysis of TfR1 expression on HMCs untreated (white layer) or treated with TfR1 miRNA (dotted layer). The histogram shows one representative experiment of six. The gray layers correspond to the isotypic-matched controls. (F) Binding of sCD89 to TfR1-His by ELISA using biotinylated sCD89. 10 µg/ml sTfR1 was coated in ELISA plates and incubated with the indicated doses of biotinylated sCD89. β-Synuclein is a His-tagged protein used as a negative control and A3 an anti-CD89 mAb as a positive control. n = 4 experiments. (G) sCD89 interaction with recombinant sTfR1. sCD89 was loaded on a column of nickel beads preincubated or not with sTfR1-His. Eluates were analyzed by SDS–10% PAGE followed by Western blot using a cocktail of anti-CD89 antibodies. (H) Flow cytometry analysis of TfR1 expression on HMCs unstimulated (gray layer) or stimulated with sCD89 (white layer). The histogram shows one representative experiment of six. (I) mRNA levels of TfR1 normalized to GAPDH mRNA levels in HMCs stimulated with sCD89 or BSA. (A–C, F, and I) Error bars indicate SEM. *, P < 0.05 (unpaired Student’s t test); **, P < 0.01; and ***, P < 0.001 (Mann-Whitney U test).
Figure 5.

sCD89 interacts with TfR1 and induces up-regulation of its expression on mesangial cells. (A–C) Frozen kidney sections immunostained for mouse TfR1 from 12-wk-old WT, α1KI, and α1KI-CD89Tg mice (A), 6-wk-old α1KI mice injected i.v. with BSA or sCD89 (B), and NOD.SCID mice injected with BSA or sCD89 (C). Arrows indicate TfR1+ cells. The corresponding graphs represent the numbers of positive cells per glomerulus counted in 20 randomly chosen fields for each mouse at 200 magnification. n = 3 mice per group. Bars: (A) 10 µm; (B and C) 5 µm. (D) Flow cytometry histogram (one representative experiment of six) of HMCs binding sCD89 (white layer) and stained with anti-CD89. The dashed layer represents incubation with sTfR1. The gray layer corresponds to the isotypic-matched control. The inset graph shows the mean binding values of indicated doses of sCD89 (±SEM) to HMCs as a ratio to background mean fluorescence intensity of seven independent experiments. (E) Flow cytometry analysis of sCD89 binding on HMCs untreated (white layer) or treated with TfR1 miRNA (dotted layer). The inset shows flow cytometry analysis of TfR1 expression on HMCs untreated (white layer) or treated with TfR1 miRNA (dotted layer). The histogram shows one representative experiment of six. The gray layers correspond to the isotypic-matched controls. (F) Binding of sCD89 to TfR1-His by ELISA using biotinylated sCD89. 10 µg/ml sTfR1 was coated in ELISA plates and incubated with the indicated doses of biotinylated sCD89. β-Synuclein is a His-tagged protein used as a negative control and A3 an anti-CD89 mAb as a positive control. n = 4 experiments. (G) sCD89 interaction with recombinant sTfR1. sCD89 was loaded on a column of nickel beads preincubated or not with sTfR1-His. Eluates were analyzed by SDS–10% PAGE followed by Western blot using a cocktail of anti-CD89 antibodies. (H) Flow cytometry analysis of TfR1 expression on HMCs unstimulated (gray layer) or stimulated with sCD89 (white layer). The histogram shows one representative experiment of six. (I) mRNA levels of TfR1 normalized to GAPDH mRNA levels in HMCs stimulated with sCD89 or BSA. (A–C, F, and I) Error bars indicate SEM. *, P < 0.05 (unpaired Student’s t test); **, P < 0.01; and ***, P < 0.001 (Mann-Whitney U test).

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