Co-targeting RANK pathway treats and prevents acquired resistance to CDK4/6 inhibitors in luminal breast cancer. Gomes et al.
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Specific primary antibodies were used overnight at the following dilution conditions: rabbit monoclonal anti phospho-Rb (Ser807/811) (D20B12), , anti phospho-NF-kB p65 (Ser536) (93H1), anti NF-kB p65 (D14E12), anti p44/42 MAPK (Erk1/2) (137F5), anti CDK4 (D9G3E), anti cyclin D1 (92G2), anti CDK2 (78B2), anti p27 Kip1 (D69C12), anti p21 Waf1/Cip1 (12D1), anti phospho-STAT1 (Tyr701) (D4A7), anti phospho-STAT1 (Ser727) (D4A7), anti STAT1 (D1K97), all fromCell Signaling, 1:1000 in 5% BSA; rabbit polyclonal anti phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (Cell Signaling), anti CDK7 (Cell Signaling) and anti phospho CDK4 (Thr172) (St John’s Laboratory), 1:1000 in 5% BSA; rabbit polyclonal anti RANK (Cell Signaling), 1:750 in 2.5% BSA; mouse monoclonal anti CDK6 (DCS83) (Cell Signaling), 1:1000 in 5% BSA; mouse monoclonal anti cE (HE12) (Sigma-Aldrich), 1:1000 in 5% Milk; and mouse monoclonal anti β-Actin antibody (#ab6276; Abcam), 1:24000 in 5% Milk. Horseradish peroxidase-conjugated (HRP) specific secondary antibodies anti-mouse-HRP IgG and anti-rabbit-HRP IgG (both from Cell Signaling) were used. Proteins were detected using the Novex® ECL HRP chemiluminescent substrate reagent kit (Invitrogen) according to the manufacturer‘s instructions, and membranes were visualized in Amersham™ Imager 680 (GE Healthcare Life Sciences). Proteins were detected by chemiluminescence.