Hrp conjugated donkey anti rabbit igg

Prior to treatment with TGF-β1 (4 ng/mL, R&D), BMP-2 (50 ng/mL, R&D), FGF-2 (20 ng/mL, Stemgent) or SB431542 (10 µM, Tocris) for the indicated time, cells were starved overnight in α-MEM containing 0.2% FCS. After stimulation and two washes with ice cold PBS, cells were harvested in RIPA lysis buffer (Millipore, Billerica, MA, USA) with protease and phosphatase inhibitors. The protein concentrations of the cell lysates were determined by the Bradford method (Bio-Rad, Hercules, CA, USA). Primary antibodies included anti-phospho-Erk, anti-phospho-p38, anti-Erk1, anti-p38, and anti-phospho-Smad3 (1:2000, Cell Signaling, Danvers, MA, USA); anti-Smad2/3 (1:2000, BD Biosciences, San Jose, CA, USA); anti-BMP type I receptor, anti-BMP type II receptor, anti-TGF-β type I receptor, and anti-TGF-β type II receptor (1:500, Santa Cruz, Santa Cruz, CA, USA); anti-collagen I (Millipore); and anti-β-actin (1:5000, Sigma-Aldrich). Secondary antibodies included horseradish peroxidase (HRP)-conjugated sheep anti-mouse IgG and HRP-conjugated donkey anti-rabbit IgG (1:5000, Cell Signaling).

The three hiPSC lines were incubated with or without BMP4 (50 ng/mL) for 4 hours. Protein was extracted as previously described [16 (link)] and concentration was determined by BCA assay (Pierce). Fifteen μg of protein was separated by SDS- PAGE (Invitrogen) and transferred to Hybond-P PVDF membrane (Amersham). The membrane was blocked with 5% non-fat dried milk in TBST for 30 minutes, incubated with diluted PhosphoSmad1/Smad5/Smad8 antibody (9511S, Cell Signaling Technology) in 5%(w/v) BSA, 1xTBST at 4°C with gentle shaking overnight, then incubated with HRP-conjugated donkey anti-rabbit IgG (1:2,000 in TBST, Cell Signaling Technology) at ambient temperature for 1 hour. Reactive protein bands were visualized using ECL plus Western blotting detection reagents (GE Healthcare) and band intensities were calculated using the Bio Rad Chemi Doc XRS imaging system. Beta-actin (13E5) Rabbit mAB (4970, Cell Signaling Technology) was used for a loading control.

BMDMs were treated as indicated, and cell lysates were subjected to Western blot. The antibodies used in this study were rabbit anti-phospho-NF-κB p65 (1:2000), rabbit anti-NF-κB p65 (1:4000), rabbit anti-phospho-Akt (1:2000), rabbit anti-Akt (Ser473) (1:4000), rabbit anti-phospho-p38 (1:2000), rabbit anti-p38 (1:4000), rabbit anti-phospho-ERK1/2 (p42/44) (1:2000), rabbit anti-ERK1/2 (p42/44) (1:4000), rabbit anti-phospho-SAPK/JNK (1:2000), rabbit anti-SAPK/JNK (1:4000), and mouse anti-β-actin (1:10000), HRP-conjugated donkey anti-rabbit IgG and HRP-conjugated sheep anti-mouse IgG (1:4000) (all from Cell Signaling Technology, Danvers, MA, USA). The signals were detected by chemiluminescence. In order to quantify the band densities among different samples, Western blot images were subjected to analysis by ImageJ software.