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. 2019 May 28;10:1164. doi: 10.3389/fimmu.2019.01164

Figure 1.

Figure 1

MAP kinases and the AP-1 complex are differentially activated by OSM and LIF. (A) Mouse calvarial osteoblasts were cultured in the absence (Co) or presence of LIF or OSM (both at 100 ng/mL) for 15 and 30 min followed by cell lysis and Western blot analysis of total, as well as phosphorylated, ERK, JNK and p38. Actin served as the internal control for protein loading. (B) Semi-quantitative PCR analysis of AP-1 subunit mRNA expression after incubation without (Co) or with LIF or OSM (both at 100 ng/mL) for 48 h. Gapdh served as loading control. (C) EMSA analysis of nuclear extracts from cells incubated for 30 min in the absence (Co) or the presence of LIF or OSM (both at 100 ng/mL). Left upper panel, EMSA for nuclear extracts incubated with AP-1 consensus probe. Middle upper panel, competition studies on nuclear extracts from OSM-stimulated cells. From left: No competitor (–), homologous unlabelled (cold) AP-1 consensus probe (C), mutated AP-1 consensus probe (M), non-homologous probe (NF-κB; NF). Right upper panel, supershifts using antibodies against c-Jun (Jun), c-Fos (Fos), and unspecific IgG (IgG) of nuclear extracts from OSM-stimulated cells. Lower panel, EMSA for nuclear extracts incubated with NF-κB consensus probe.