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. 2013 May 31;191(1):369–377. doi: 10.4049/jimmunol.1201864

FIGURE 3.

FIGURE 3.

ALCAM is expressed on MAECs and binds S100B, leading to an ALCAM-dependent NF-κB activation in RAGE−/− MAECs. (A) RAGE−/− MAECs express ALCAM mRNA as shown by PCR in lane 1 (results of three different cell populations 1–3 are shown) and ALCAM Ag as shown by Western blot in lane 2 (results of four different cell populations a–d are shown). (B) RAGE−/− MAECs incubated with 800 nM S100B for 6 h were cross-linked (control: no cross-linking) and harvested. Next, immunoprecipitation of protein lysate was performed with anti-S100B as described in Materials and Methods. The IP material was separated and stained with anti-ALCAM Ab (1:600). (C) Recombinant ALCAM was separated using electrophoresis, transferred to a membrane, and renatured. Incubation with S100B overnight was followed by washing and anti-S100B staining as described in Materials and Methods. (D) S100B treatment for 6 h leads to a dose-dependent increase of NF-κB–binding activity in RAGE−/− MAECs, determined by EMSA. Densitometry normalized to consensus (background). (E) Time course of NF-κB–binding activity from MAECs incubated with 800 nM S100B. Time points are indicated on the x-axis. Lane 8 contains specificity control with unlabeled consensus NF-κB oligonucleotide. The experiment was repeated two times, and EMSA of one representative experiment shown. Densitometry of two experiments, normalized to consensus (background). (F) ALCAM silencing in RAGE−/− MAECs leads to highly efficient suppression of ALCAM protein and mRNA expression 2 d after transfection, as shown in Western blot and PCR. (G) ALCAM silencing in RAGE−/− MAEC 2 d prior to treatment with 800 nM S100B (for time points as indicated on x-axis) leads to 90% inhibition of NF-κB–binding activity. The experiment was repeated two times, and EMSA of one representative experiment is shown. Densitometry of two experiments, normalized to consensus (background). (H) S100B treatment (800 nM) at optimal time point of 6 h on NF-κB–binding activity of MAEC RAGE−/− with and without ALCAM silencing. Data are the mean percentage ± SE from three independent experiments. *p < 0.01, **p < 0.009. (I) Supershift (anti-p50, anti-p65, anti-cREL, anti-RelB, or anti-p52 Abs) from RAGE−/− MAECs treated with 800 nM S100B for 6 h. Lanes: 1, control; 2, S100B treated; 3–7, S100B treated (Abs as specified); 8, consensus oligo. (J) Supershift as in (I), but with prior ALCAM silencing. (K) Western blot for different NF-κB subunits with 10 μg of nuclear extract from RAGE−/− MAECs after 6 h of 800 nM S100B with (2) or without (3) prior silencing of ALCAM. Lane 1, control without S100B.