2.3. Western blots:

O9-1, MC3T3-E1, MMPCs and HBO cell lines was seeded in 6-well plates (Costar, 3516) and cultured until 100% confluency. The cells were then serum starved for 12 hours prior to treatments. The cells were first treated with 5μM WP1066 (inhibitor of JAK2 phosphorylation) (Millipore, S65784-10mg) and 15μM DAPT (a γ-secretase inhibitor) (Sigma, D5942-25mg) for 1 hour prior to stimulus with: JAG1-Fc-Beads complex (10μg), Fc-Beads complex (10μg), BMP2 (100ng/ml), 10ng/ml IFN-γ (activator of JAK2 phosphorylatiion), 5μM WP1066 and 15μM DAPT. Cells were then lysed using RIPA buffer (Thermoscientific, 89900) containing a protease inhibitor (Roche, 05892791001) and a phosphatase inhibitor (Roche, 04906845001) to obtain whole cell proteins that were resolved on an 8% SDS-PAGE gel and then transferred to a nitrocellulose membrane. The blots were then placed in 1.5% BSA (Sigma, A2153-100G) + 0.1% Tween-20 for 30 minutes and thereafter incubated at 4°C for 16 hours with a primary antibody against phosphorylated JAK2 (Cell signaling, 8082S, AB_10949104, Mol.wt 125KDa) and later total-JAK2 (Cell signaling, 3230S, AB_2128522, Mol.wt 125KDa) at 1:1000 dilution in 1.5% BSA + 0.1% Tween-20. The blots were moved to secondary antibody (Cell signaling, 7074P2) prepared at a 1:3000 dilution in 1.5% BSA + 0.1% Tween-20 and incubated at room temperature for 1 hour, then washed using TBS (Amresco, 0307-5L) + 0.1% Tween-20 3 times, 5 minutes each wash. Blots were developed by chemiluminescence using the Bio-rad Chemidoc MP imaging system available at the Emory Children’s Center Pediatric research equipment core.