P38 mapk

For protein analyses of lung tumor cell lines, cells were harvested, washed in PBS, and resuspended in cell lysis buffer (9803, NEB) supplemented with protease inhibitor cocktail (MilliporeSigma). Protein amount was measured by Lowry assay. The following antibodies were used for Western blot analyses: c-Jun (610326, BD Biosciences), phospho–c-Jun Ser63 (9261, New England Biolabs [NEB]), phospho–c-Jun Ser73 (9164, NEB), JunD (sc74, Santa Cruz Biotechnology Inc.), JunD (5000, NEB), phospho–SAPK/JNK Thr183/Tyr185 (4668 and 9251, NEB), SAPK/JNK (9252, NEB), phospho–p38 MAPK Thr180/Tyr182 (9211, NEB), p38 MAPK (9212, NEB), tubulin (ab7291, Abcam), and β-actin (ab49900, Abcam). All primary antibodies were used at 1:1000 dilution. Horseradish peroxidase–conjugated (HRP-conjugated) secondary antibodies were used at 1:5000 dilution, and anti–β-actin HRP antibody was used at 1:30,000 dilution. For Western blot utilizing the LI-COR system, DyLight goat anti–mouse 680 (5470, NEB) and DyLight goat anti–rabbit 800 (5151, NEB) secondary antibodies were used at 1:15,000 dilution. Separate immunoblots were used for each antibody.

Protein samples were boiled and run on SDS-PAGE gels, followed by electrophoretically transferring onto nitrocellulose membranes. The nitrocellulose membranes were then blocked with 5% nonfat dried milk in TBST buffer. The blocked membranes were incubated with an antibody to glial fibrillary acidic protein (GFAP) (1 : 4000; Millipore Corp., MA, USA), synaptophysin (SYP) (1 : 4000, Abcam, Cambridge, United Kingdom), p38 MAPK (1 : 1000; New England Biolabs, Beverly, MA), phospho-p38 MAPK (1 : 1000; New England Biolabs, Beverly, MA), and p-Stat3 (1 : 1000; Cell Signaling Technology, Danvers, MA) and Stat 3 (1 : 1000; Cell Signaling Technology, Danvers, MA) in TBST milk overnight at 4°C. We used β-actin as a loading control (1 : 5000; Santa Cruz Biotechnology Inc., CA, USA). Values used for statistical analysis were expressed as the ratio of the band of each protein to the band of their loading control.

Proteins (20 µg) were separated by SDS-polyacrylamide gel electrophoresis and blotted to PVDF membranes (Bio-Rad, Munich, Germany). Membranes were incubated with antibodies in 1.5% BSA/PBS/0.1% Tween 20. Signals were detected with the ECL Western blot detection system (Amersham Pharmacia, Deisenhofen, Germany). E-cadherin, ERK1/2, GAPDH and p38 MAPK antibodies were from New England Biolabs GmbH (Frankfurt, Germany). Antibodies to detect human and murine chemerin were from R&D Systems (Wiesbaden-Nordenstadt, Germany). ImageJ software was used for quantification [48 (link)].