Phospho jnk

Proteins were extracted from human and mice myometrium using RIPA buffer. The protein concentrations were determined using the BCA Protein Assay Kit (23227, Pierce). A total of 50 μg of protein samples was resolved on a 10% SDS-PAGE gel. Nitrocellulose membranes were then incubated overnight at 4°C with specific primary antibodies: TLR4 (AF7017, Affinity), ERK (BF8004, Affinity), Phospho-ERK (BF8011, Affinity), OTR (AF4667, Affinity), CX-43 (BF8212, Affinity), JNK (AF6318, Affinity), Phospho-JNK (AF3318, Affinity), P65 (AF5006, Affinity), Phospho-P65 (AF2006, Affinity), P38 (8690S, Cell Signaling), and Phospho-P38 (4511S, Cell Signaling), all at a dilution of 1:1000. Subsequently, the nitrocellulose membranes were incubated with secondary fluorescent antibodies at a dilution of 1:5000 for 1 h at room temperature. Band densitometry was analyzed using Quantity One imaging software (Bio-Rad, Hercules, CA, USA).

All proteins were collected from cells or lung tissues as reported previously (Ning et al., 2004 (link)). The monoclonal antibodies used in this study were: α-SMA (Affinity, BF9212), Collagen Ⅰ (Affinity, AF7001), Fibronectin (Affinity, AF5335), β-tubulin (Affinity, T0023), GAPDH (Affinity, AF7021), Phospho-Smad3 (Affinity, AF3362), Smad3 (Affinity AF6323), Smad2 (Affinity, AF6449), Phospho-Smad2 (Affinity, AF3450 Phospho-p38 (Affinity, AF4001), p38 (Affinity, BF8015), Phospho-JNK (Affinity, AF3318), JNK (Affinity, AF6318), Phospho-ERK (Affinity, AF1015), ERK (Affinity, AF0155), Phospho-AKT (Affinity, AF0832), AKT (Affinity, AF6212), Cleaved PARP (Affinity, AF7023), E-cadherin (Cell Signaling Technology, 14472S), N-cadherin (Cell Signaling Technology, 13,116), Vimentin (Cell Signaling Technology, 5,741). The monoclonal antibodies are diluted 1:1,000 with skimmed milk powder.