Goat anti rabbit igg hrp

Anti-CPSF5 (1:5000; 10322-1-AP, Proteintech), anti-CPSF6 (1:5000; ab175237, Abcam), anti-CPSF7 (1:2000; 55195-1-AP, Proteintech), anti-RIG-I (1:1000; D14G6, Cell Signaling), anti-MDA5 (1:1000; D74E4, Cell Signaling), anti-IFIT3 (1:2000; 15201-1-AP, Proteintech), anti-ISG15 (1:2000; 15981-1-AP, Proteintech), anti-DDX21 (1:2000; 10528-1-AP, Proteintech), anti-DHX15 (1:2000; 12265-1-AP, Proteintech), anti-CCL2 (1:2000; 66272-1-Ig, Proteintech), anti-IRF3 (1:1,000; sc-33641, Santa Cruz), anti-phosphorylated IRF3 (1:1000; 4D4G, Cell Signaling), anti-TBK1 (1:1000; D1B4, Cell Signaling), anti-phosphorylated TBK1 (1:1000; D52C2, Cell Signaling), anti-VSVG (1:1000; V5507, Sigma), anti-ICP27 (1:1000; ab31631, Abcam), anti-GAPDH (1:20000; 60004-1-Ig, Proteintech), anti-HA (1:5000; clone HA-7, H6533, Sigma), anti-Flag (1:5000; clone M2, F1804, Sigma), Anti-rabbit IgG (H+L), (Alexa Fluor 555 Conjugate) (1:1000, 4413, Cell Signaling), goat anti-mouse IgG-HRP (1:10000; HA1006, HuaBio), goat anti-rabbit IgG-HRP (1:10000; HA1001, HuaBio). The poly(I:C) (LMW) and poly(dA:dT) were purchased from InvivoGen. Lipofectamine 3000 and Lipofectamine RNAiMAX were purchased from Invitrogen. Act D (actinomycin D) was purchased from Sigma.

After proper treatment, the cells and tissues were lysed in RIPA buffer (Sigma, USA) with a protease inhibitor and phosphatase inhibitor cocktail. The same amounts of protein samples were separated by SDS–PAGE and transferred onto a polyvinylidene fluoride membrane. After blocking with 10% milk or 3% BSA, the membranes were incubated with primary antibodies overnight at 4 °C and then with horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h at room temperature. Concentrations adopted for the above-mentioned antibodies were based on the manufacturers’ specifications. Finally, the membranes were observed using an enhanced chemi-luminescence kit (Millipore, Bedford, MA). Blot intensity was analyzed with Quantity One. The details for the primary antibodies were as follows: Parkin(1:1000, Cat: ab15954, Abcam), p-STAT3 (1:100000, Cat: ab76315, Abcam), Caspase-3 (1:1000, Cat: 9662 s, Cst), Bcl-2 (1:1000, Cat: 15071 s, Cst), STAT3 (1:1000, Cat: 12640 s, Cst), p-JAK2 (1:1000, Cat: 3776 s, Cst), VEGF (1:1000, Cat: RT1649, Huabio), and GAPDH (Cat: RT1210-1, Huabio). The secondary antibodies were as follows: goat anti-rabbit IgG–HRP (1:2000, Cat: HA1001-100, Huabio) and goat anti-mouse IgG–HRP (1:2000, Cat: HA1006, Huabio).

Cells were lysed with the mixture containing cell lysis buffer for Western and IP and 1% phenylmethanesulfonyl fluoride (PMSF) (Biosharp, Beijing, China) on ice to extract protein. Protein samples were separated by SDS-PAGE and then transferred to polyvinylidene difluoride membranes. And the membranes were blocked with 5% non-fat milk at room temperature for 1 h, then incubated with the primary antibody overnight at 4 °C and next with the secondary antibody for 1 h at room temperature. The protein bands were visualized using ECL Protect from Light (Biosharp) and quantified using Image J software. The primary antibodies used in this study were as follows: ADIPOQ (sc-136131, Santa Cruz, Watsonville, CA, USA, diluted 1:200), FLAG (20543–1-AP, Proteintech, Rosemont, IL, USA, diluted 1:1,000), YTHDF1 (17479–1-AP, Proteintech, diluted 1:1,000), GFP (ET160–25, Huabio, Hangzhou, China, diluted 1:5,000), β-actin (M1210–2, Huabio, diluted 1:5,000). The secondary antibodies were as follows: goat anti-mouse IgG-HRP (HA1006, Huabio, diluted 1:2,000), goat anti-rabbit IgG-HRP (HA1001, Huabio, diluted 1:2,000).