Tgx pre cast gels

Immunopurified proteins were clicked on beads by a 30-min incubation at 20 °C with vigorous intermittent shaking in 200 µL of IPD buffer containing 4 mM CuSO₄, 5 µM azido-AlexaFluor647 and 10 mM sodium ascorbate. The beads were washed with IPD buffer and resuspended in 20 µL SDS-Lysis Buffer to which 20 µL of SDS Loading buffer was added. Beads were incubated 5 min at 95 °C in this solution and 20 µL of supernatant was analyzed on gradient gels (BioRad 4–12% TGX pre-cast gels). The modified proteins were detected with an infrared imager (Odyssey, LI-COR Bioscience) after transfer onto a nitrocellulose membrane (Protran, 45 µm pores, GE Healthcare) and total proteins on the membrane were stained with Ponceau S.

Mouse VEGF-A ELISA (Biosource, Invitrogen) was performed following manufacturer's instructions. ACM were isolated as described (see above) from WT sham and TAC mice and immediately collected on RIPA lysis buffer. Western blots were performed as described34 (link). Briefly, samples were loaded and run on a 4–12% TGX-precast gels (Bio Rad) after being heated on 2 × Laemmli sample buffer with β-mercaptoethanol (Bio Rad) for 10 min at 70 °C. proteins were then transferred using the Trans-Blot Turbo Transfer System (Bio Rad) into PVDF membranes that were blocked with 5% Difco skin milk (BD Bioscience) before antibody incubations. Antibodies used were rat primary anti-RBPJ (1:500) antibody (generously provided by Professor T. Honjo, Kyoto University30 (link)), ANF (1:500) (Millipore, AB5490) and GAPDH (1:2,000) (Abcam, ab9485).