Anti phospho jnk p jnk

Samples were lysed in lysis buffer containing 50 mmol/L Tris–HCl (pH 6.8) and 10% sodium dodecyl sulfate, and the protein concentration of each sample was determined using a Bio‐Rad Protein Assay kit (Bio‐Rad Laboratories). Samples were heated at 95°C for 5 min and subjected to electrophoresis using SuperSep Ace 10%–20% gels (Fujifilm) at 20 mA for 80 min. The Trans‐Blot Turbo Transfer System (Bio‐Rad) was used to transfer proteins onto a polyvinylidene fluoride membrane (Bio‐Rad). The membrane was incubated consecutively in iBind solution (Invitrogen) containing primary and secondary antibodies. Primary antibodies included anti‐TNF‐α (rabbit monoclonal, dilution 1:400, #11948; Cell Signaling Technology), anti‐iNOS (rabbit monoclonal, dilution 1:400, #13120; Cell Signaling Technology), anti‐ MMP‐9 (rabbit polyclonal, dilution 1:1000, #ab38898; abcam), anti‐ MMP‐2 (rabbit polyclonal, dilution 1:1000, #ab97779; abcam), anti‐IL1RL2 (rabbit polyclonal, dilution 1:1000, # PA5‐38013; Invitrogen), anti‐p38 (rabbit polyclonal, dilution 1:5000, #9212; Cell signaling technology), anti‐phospho‐p38 (p‐p38) (rabbit polyclonal, dilution 1:1000, #9211; Cell signaling technology), anti‐JNK (rabbit polyclonal, dilution 1:5000, #9252; Cell signaling technology) and anti‐phospho‐JNK (p‐JNK) (rabbit polyclonal, dilution 1:1000, #9251; Cell signaling technology).