Goat anti mouse igg

Western blot was used to semiquantitatively detect the protein expression of Angptl 2 in lung tissues. Total proteins were extracted, and the protein concentration was measured. After balancing the concentration, the proteins were separated by gel electrophoresis and electrotransferred to a nitrocellulose membrane. Following blocking, the membrane was conjugated with the primary antibody (goat anti-mouse IgG, R&D Systems, Inc.) in a blocking solution, incubated with a secondary antibody (horseradish peroxidase-labeled Hag Anti Goat IgG) at room temperature, and illuminated with a chemiluminescence reaction kit in a dark room. The optical density of each film strip was analyzed using the Leica Q-5501W image analysis system [25 (link)].

Nuclear, cytoplasmic (CYT) and plasma membrane (PM) protein of PFC tissues or astrocytes were isolated by commercial kits (KGP1100, KeyGEN BioTECH; P0033, Beyotime Biotechnology). Phenylmethanesulfonyl fluoride (PMSF, a protease inhibitor) was used to inhibit protein degradation. The protein concentration of sample was detected by bicinchoninic acid (BCA) protein assay kit (Thermo scientific). Lysates were loaded into 10% sodium dodecyl sulfate-polyacrylamid gel electrophoresis and subsequently transferred to polyvinylidene fluoride membranes. Membranes were blocked in 5% non-fat milk for 1 h. Then, the following first antibodies were used: GLUT1 (1:5000, ab115730, Abcam), TXNIP (1:1000, #14715, CST), Na, K ATPase (1:1000, #3010, CST), GR (1:1000, #3600, CST), Histone H3 (1:500, sc-517576, Santa Cruz) and β-actin (1:2000, #4970, CST). After incubating with first antibody overnight, membranes were incubated goat anti-mouse IgG (1:5000, HAF007, R&D systems) or goat anti-rabbit IgG (1:3000, #7074, CST) for 1.5 h. Protein bands were visualized by ECL (#180–5001, Tanon) and imaged by chemiluminescence imaging instrument (#5200, Tanon). The gray value of images was measured by ImageJ.

Grafts and SPCs were lysed in fresh extraction buffer (Sigma-Aldrich, St. Louis, MO, USA) supplemented with a protease inhibitor and phosphatase inhibitor (Gold Biotechnology, St. Louis, MO, USA). The extracted proteins were separated on a 15% SDS-polyacrylamide gel and electroblotted onto a polyvinylidene fluoride membrane. The membrane was blocked using 5% skim milk-containing TBST for 60 min at 20–25°C and incubated with primary antibodies against Bcl-2 (Affinity), Bax (Affinity), cytochrome c (Affinity), cleaved-caspase-3(Affinity), cleaved-PARP (Affinity), and PCNA (Abcam) overnight at 4℃. The membranes were then washed with TBST and incubated with goat anti-mouse IgG (R&D Systems, Inc., Minneapolis, MN, USA) and goat anti-rabbit IgG (R&D Systems) secondary antibodies. Bound antibodies were detected using an electrochemiluminescence detection system (Amersham Life Science, Arlington Heights, IL, USA). β-actin was used as the loading control.