Mice were perfused transcardially with 40 ml of cold PBS. Hemispheres were isolated and stored at −80 °C until analysis. Protein extraction and western blots were performed as previously described52 (link). Briefly, the whole-cell lysates were obtained by homogenizing in RIPA lysis buffer (Santa Cruz Biotechnology, Inc., sc-24948) and centrifuging (14,000 g at 4 °C for 30 min). Equal amounts of protein (50 mg) were loaded and subjected to electrophoresis on an SDS-PAGE gel. After being electrophoresed and transferred to a nitrocellulose membrane. To separate the region where the target protein will appear, the membrane was cut along the molecular weight marker. The member straps were then blocked and incubated with the primary antibody overnight at 4 °C. Following primary antibodies were used: anti-tryptase 1:1000 (Santa Cruz Biotechnology, sc-32889), anti-chymase 1:100 (Abcam, ab186417), anti- IL-1β (Abcam, ab9722) 1:750 and anti SHIP1 1:500 (Santa Cruz Biotechnology, sc-6244). The antibody against β-actin (Santa Cruz, 1:1000) was used as the internal control.
If the target protein had similar (+/− 20 kDa) weight compared to β-actin, the membrane straps were blocked and proceeded as described above.
Some representative strips were proceeded with “Microsoft Office 2010”
If the target protein had similar (+/− 20 kDa) weight compared to β-actin, the membrane straps were blocked and proceeded as described above.
Some representative strips were proceeded with “Microsoft Office 2010”