Flag and β actin

BMMs and/or pre-OC (BMMs treated with RANKL for 2 days) were lysed in cell lysis buffer (Cell Signaling Technology, Danvers, MA, USA) post treatments. Protein concentration was determined using BCA (Pierce, Invitrogen) and equal amounts of protein was loaded onto SDS-PAGE. After transfer, and blocking in 5% BSA for 1 hr at room temperature, membranes were probed with primary antibodies in 5% BSA in PBS-Tween (1% v/v) for overnight and then washed with PBS-Tween (3x) and probed with secondary antibodies from LI-COR (Odyssey Imager; donkey anti-rabbit and anti-mouse) for 1 hr at room temperature. Membranes were then with PBST (3x) and scanned by using LI-COR Odyssey Imager (LI-COR Biosciences, Lincoln, NE, USA). Western blots were also performed (for LC3 and actin) using capillary-based immunoassay using the Wes-Simple Western method with the anti-rabbit detection module (Protein Simple). Protein expression was measured by chemiluminescence. The NEMO and ISG15 antibody were purchased from Santa Cruz, Dallas, TX, USA; phos-p65, p65 and LC3 antibodies were purchased from Cell Signaling Technology, Danvers, MA, USA; Flag and β-actin was purchased from Sigma, St. Louis, MO, USA.

Two days after plasmid transfection, cells were harvested and centrifuged, suspended in 500 μl of NET-2 buffer [50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM phenylmethanesulfonylfluoride, 2 mM benzamidine, 0.05% NP-40] containing 100 U of RNase inhibitor (Fermentas) and sonicated using a Branson Sonifier 250. After centrifugation, the supernatant was subjected to IP as previously described (3 (link),25 (link),30 (link)). Where indicated, the supernatant was treated with 2 μg of RNase A (Sigma) before IP and incubated for 15 min at 37°C. When indicated, cells were incubated with 1% of formaldehyde (Sigma-Aldrich) in phosphate-buffered saline for 10 min before harvesting (38 (link)).
The following antibodies were used for western blotting: FLAG and β-actin (Sigma-Aldrich), Myc (Calbiochem), HA (Roche), CBP80 (4 (link)), eIF4E, eIF3a, and phospho-S/TQ (Cell Signaling Technologies), eIF3b and eIF3c (Santa Cruz Biotechnology), eIF4GI (a gift from S. K. Jang), CTIF (3 (link)), SLBP (39 (link)), SLIP1 (40 (link)), UPF1 (a gift from L. E. Maquat), PNRC2 and DCP1A (25 (link)), SMG5 (Abcam), SMG6 (a gift from S. Ohno), SMG7 (Bethyl), GST (Amersham) and 6xHis (GE Healthcare).