Anti pstat4

Standard Western-blot assays were used as described previously^{59 (link)}. The following primary antibodies were used: anti-PPARα (Abcam, Cata# ab24509), anti-ACLY (Santa Cruz, Cata# sc-517267), anti-FASN (Santa Cruz, Cata# sc-48357), anti-ACSL1 (cell signaling, Cata# 4047 S), anti-ACSL5 (Santa Cruz, Cata# sc-365478), anti-STAT3 (Santa Cruz, Cata# sc-8019), anti-pSTAT3 (cell signaling, Cata# 9145 S), anti-STAT1 (cell signaling, Cata# 14994 S), anti-pSTAT1 (cell signaling, Cata# 9177 S), anti-STAT4 (cell signaling, Cata# 2653 S), anti-pSTAT4 (cell signaling, Cata# 4134 S), anti-AKT (Santa Cruz, Cata# sc-5298, 1:2000 dilution), anti-pAKT (cell signaling, Cata# 9018 S), anti-pERK (cell signaling, Cata# 4376), anti-ERK (cell signaling, Cata# 9102), anti-pMAPK (cell signaling, Cata# 4511), anti-MAPK (cell signaling, Cata# 9212) and anti-β-actin (Sigma, Cata# A5441, 1:100,000 dilution) antibodies. Other than anti-AKT and anti-β-actin antibodies, the dilution for all other antibodies was 1:1000.

Total protein was extracted using RIPA buffer containing a cocktail of proteinase and phosphatase inhibitors (Sigma-Aldrich). Protein concentration was measured using BCA kit (Thermo Scientific, Pierce) and 30 μg/lane of total protein was loaded onto 10% polyacrylamide gels. The membranes were first incubated with following anti-phosphotyrosine antibodies at 4°C overnight: anti-pJAK2 (Cell Signaling Technology, Cat#3771, dilute 1:1000) and anti-pSTAT4 (Santa Cruz, Cat#sc-28296, dilute 1:100). The membranes were subsequently stripped using a solution containing 100 mM β-mercaptoethanol, 2% SDS, and 62.5 mM Tris-HCl (pH 7.6) at 50°C for 30 min, and re-incubated with anti-JAK2 (Cell Signaling Technology, Cat#3230, dilute 1:1000) or anti-STAT4 (Cell Signaling Technology, Cat#2653, dilute 1:1000) antibodies at 4°C overnight.

EBV-B cells were incubated with 10, 20 or 40 μM LSF (Sigma-Aldrich) for 2, 4 or 16 h after which cells were collected and processed for quantitative RT-PCR or immunoblot analysis. Stimulation with IL-12 was performed following 2 h treatment of EBV-B cells with 20 μM LSF using 2 ng/ml IL-12 (Immunotools, Friesoythe, Germany) for 1 or 2 h.
For immunoblot analyses, cells were lysed in 1% Triton X-100 in 20mM Tris-HCl (pH 8) and 150 mM NaCl (in the presence of a protease inhibitor cocktail, Calbiochem, San Diego, CA). Immunoblot analysis was carried out by chemiluminescence (SuperSignal West Pico Chemiluminescent Substrate kit, Pierce, Waltham, MA) using as primary Abs anti-ShcA (Upstate Biotechnology, Lake Placid, NY), anti-STAT4 (Cell Signaling, Danvers, MA), anti p-STAT4 (Santa Cruz, Santa Cruz, CA), anti-actin (Millipore, Billerica, MA) or anti-Lamin A/C (Cell Signaling) Abs and secondary peroxidase-labeled Abs (Amersham Pharmacia Biotech, Uppsala, Sweden).