Experiments were performed previously as described [37 (link), 39 (link)]. Cells were lysed using RIPA buffer and protein was separated by SDS-PAGE. Protein (50 µg) levels were quantified using antibodies raised against human proteins: rabbit anti-fibulin-3 (1:1000; Abcam), rabbit anti-KISS1R (1:4000, Abcam), rabbit anti-MMP-9 (1:500; Abcam), rabbit anti-ERK1/2 (1:1000; Cell Signaling), rabbit anti-phoshpo-ERK1/2 (1:2000; Cell Signaling), rabbit anti-AKT (1:1000; Cell Signaling), rabbit anti-phospho-AKT (1:1000; Cell Signaling). β-Actin or glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression was used as a loading control and was examined using rabbit anti-Actin (1:5000; GeneTex Inc.) or mouse anti-GAPDH (1:3000; GeneTex Inc.). After 1-hour incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies, rabbit (1:2500, GE Healthcare) or mouse (1:2500, GE Healthcare), the proteins were visualized using SuperSignal West Dura Extended Duration Substrate (Thermo Scientific) and a VersaDoc Imaging System (Bio-Rad).
To determine the effect of KP-10 treatment on fibulin-3 expression, cells were treated with KP-10 (100 nM) (Calbiochem) in FBS-supplemented media for 72 hours, where applicable.
To determine the effect of KP-10 treatment on fibulin-3 expression, cells were treated with KP-10 (100 nM) (Calbiochem) in FBS-supplemented media for 72 hours, where applicable.
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