α p38 mapk

Cells were lysed using lysis buffer (40 mM Tris-HCl pH 8.0, 150 mM NaCl, 1% sodium dodecyl sulfate (SDS)). Approximately 20 μg of the protein extract was resolved via SDS-PAGE and analyzed via immunoblotting with primary antibodies against α-HIF-1α (Cell Signaling, Danvers, MA, USA, 3716), α-PARP (Cell Signaling, 9542), α-Caspase-3 (Cell Signaling, 9662), α-p44/42 MAPK (ERK1/2) (Cell Signaling, 4695), α-Phospho-p44/42 MAPK (Erk1/2) (Cell Signaling, 4370), α-p38 MAPK (Cell Signaling, 9212), α-Phospho-p38 MAPK (Cell Signaling, 4511), α-JNK2 (Cell Signaling, 9258), α-Phospho-SAPK/JNK (Cell Signaling, 4668), α-Cyclin B1 (Santa Cruz, Dallas, TX, USA, sc-752), α-Cyclin D1 (Cell Signaling, 2978), α-Cyclin E1 (Cell Signaling, 4129), α-CDK4 (Cell Signaling, 12790), α-p53 (Millipore, Burlington, MA, USA, 05-224), α-p21 (Cell Signaling, 2947), α-TBP (Abcam, Cambridge, UK, ab818), α-β-actin (Santa Cruz, sc-47778), and α-alpha-tubulin (Abcam, ab7291).

Gp96, calreticulin, and HSP90 were isolated from murine livers as previously described^{10 (link),22 (link)}. The quality and purity of the gp96 preparations was vigilantly verified by SDS-PAGE gel and immunoblotting and is consistent with our previous studies.
For immunoblots, cells were lysed with NP-40 and lysates were run on SDS-PAGE gels and transferred to nitrocellulose membranes. Membranes were probed with the following antibodies $\alpha$ -STAT1(14944), $\alpha$ -pSTAT1 S727(9177), $\alpha$ -pSTAT1 Y701(9167), $\alpha$ -pp38 MAPK(9167), $\alpha$ -p38 MAPK(9216), $\alpha$ - $\mathrm{\beta }$ -actin(8457), (Cell Signaling) and $\alpha$ -SHP2(sc-7384) (Santa Cruz). Immunoblots comparing different cell types and timepoints were run with all on a singular membrane when possible. Membranes were developed using a Proteinsimple imager and quantitated using Alpha View software.
Luminex (eBioscience) consisted of the single analytes: IL-12, IL-18, IL-15/IL-15R, IFN- $\alpha$ , IP-10, and RANTES. Samples were analyzed using the manufacturer’s protocol and a Bioplex II Luminex machine.
CXCL10 and CCL5 ELISAs were purchased from eBioscience and samples were analyzed following the manufacturer’s protocol.