Veriblot hrp

Western blot was performed as previously reported (27 (link)). The following antibodies were used for immunoblotting: OX40L (14991S), p105/p50 (13681S), p65 (6956S), RelB (10544S), c-Rel (67489S), HDAC1 (5356S), HSP90 (4877S), P-STAT3 (Tyr705, 9145S), STAT3 (12640S), P-STAT6 (Tyr641, 56554S), STAT6 (5397S), β-Actin-HRP (5125S), Goat anti-Rabbit IgG-HRP (7074S), Horse anti-Mouse IgG-HRP (7076S) from Cell Signaling Technology; p100/p52 (05–361) from Millipore; StarBright Blue 700 Goat anti-Mouse IgG (12004159), StarBright Blue 520 Goat anti-Rabbit IgG (12005870) from Bio-Rad; VeriBlot-HRP (ab131366) from Abcam. Images were acquired and analyzed using Bio-Rad ChemiDoc MP Imaging System.

SDS in the tumor cytoskeleton fraction was removed with the Pierce Detergent Removal Spin Column (Thermo, 87777). For IP, 100 μg of the protein extract was diluted in 500 μL of IP buffer containing 1% bovine serum albumin (BSA; Calbiochem, 2930) and 0.1% SDS, which helps to prevent self-assembly of cytokeratin proteins. The mixture was precleared by centrifugation at 16,000 × g for 10 min, and the supernatant was transferred into a new tube. Then, 5 μg of rabbit isotype IgG (Thermo, 10500C) or rabbit anti-CXCL12 antibody (LSBio, LS-C48888) was added to the solution and incubated on a rotor at 4 °C overnight. Separately, 100 μL of protein G agarose beads (Cell Signaling, 37478; 50% slurry) were preblocked in IP buffer containing 1% BSA. After overnight incubation, 20 μL of pure protein G resins (or 40 μL of 50% slurry) was added directly to the protein/antibody solution and incubated for another 2 h at 4 °C. Following three washes with IP buffer containing 0.1% SDS, the resins were boiled in 100 μL of Laemmli sample buffer containing 20 mM DTT, and 10 μL of each sample was applied to an SDS-PAGE gel for Western blotting, where Veriblot-HRP (Abcam, ab131366) was used.