C jun 9165

Cells were plated at 25% confluency in 8-well chamber slides and treated for 48 and/or 72 hours. Media was removed, cells were washed twice with cold PBS and fixed in ice-cold methanol:acetone 1:1 for 10 minutes at −20°C. Fixative was removed and cells were washed twice more with cold PBS before incubation for 12 minutes on ice in permeabilization buffer (0.05% TritonX-100 in PBS). Cells were blocked in 5% normal goat serum in PBS for one hour at room temperature. Phospho-JNK(Thr183/Tyr185) (#9251) and c-Jun (#9165) primary antibodies were purchased from Cell Signaling (Danvers, MA) and used at dilutions of 1:300 and 1:500, respectively. After overnight incubation in primary antibody at 4°C, cells were washed and incubated with AlexaFluor568 (#A11011) at a dilution of 1:2000 for one hour at room temperature. Cells were washed three times in PBS and slides were mounted using VectaShield containing DAPI (#H1200 Vector Laboratories, Burlingame, CA). Fluorescent images were taken at 4000x or 6000x using a Leica DM4000B (Buffalo Grove, IL) upright fluorescent microscope. Leica Application Suite v3.7 (Leica, Buffalo Grove, IL) software was used to capture images. Quantification of c-Jun positive nuclei was performed manually.

Lysates were prepared from BMDMs on ice in RIPA buffer (ab156034; Abcam) containing protease and phosphatase inhibitors (78444; Thermo Fisher Scientific). Proteins were separated on Tris–HCl gradient gels (5678085 or 5678084; Bio-Rad Laboratories) and transferred to nitrocellulose (N010600001; Amersham). Membranes were blocked in 5% BSA (A2153; Sigma-Aldrich) and probed with primary antibodies overnight at 4°C. Anti-pStat6 (56554), Stat6 (5397), pJNK (9251), JNK (9252), pERK (9101), ERK (9102), p-p38 (9211), p38 (9212), Nos2 (2977), pJunB (8053), JunB (3753), p-cJun (9261), and cJun (9165) antibodies were obtained from Cell Signaling Technology. Irg1 antibody (ab222411) was purchased from Abcam. As loading control anti-Grb2 antibody (610112) was used from BD. Membranes were washed and probed with secondary antibodies at a 1:10,000 dilution and developed using chemiluminescence reagents (34580; Thermo Fisher Scientific).

Antibodies used in the study are β-actin (AC-15), flag epitope (F1804), myc (M4439), PABP (P6246) and Vinculin (V9264) from Sigma-Aldrich; TIA-1 (sc-1751) and p-c-Jun (Ser63/73, sc-16312) from Santa Cruz Biotechnology; RFP antibody (GTX59862) and Vinexin (GTX115362) from GeneTex; GFP (#29779) from AnaSpec; c-Jun (#9165) from Cell Signaling Technology and AlexaFluor 488, 594 or 647-conjugated secondary antibodies from Invitrogen. The CPEB4 monoclonal antibody was raised using the N-terminal 458 amino acids (a.a.) of rat CPEB4 (rCPEB4) produced in Escherichia coli. The CPEB2 antibody has been described elsewhere [10] (link). All chemicals were obtained from Sigma-Aldrich.