Phospho jnk1 2

Protein extraction, subcellular fractionation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and immunoblot analyses were performed according to previously published procedures [41 (link)]. Briefly, samples were separated using 7.5% gel electrophoresis and transferred to nitrocellulose membranes. The nitrocellulose membranes were then incubated with the indicated primary antibody, followed by incubation with horseradish peroxidase-conjugated secondary antibody. Immunoreactive proteins were visualized using ECL chemiluminescence detection (Amersham Biosciences, Buckinghamshire, UK). Equal protein loading was monitored, and the integrity of subcellular fractionation was verified using β-actin immunoblotting. Antibodies against iNOS and IκBα were provided by Santa Cruz Biotechnology (Dallas, TX, USA). Phospho-ERK1/2, ERK1/2, phospho-p38, p38, phospho-JNK1/2, JNK1/2, phospho-c-Jun, c-Jun, phospho-c-Fos, c-Fos, lamin, and phospho-IκBα were obtained from Cell Signaling (Danvers, MA, USA). Horseradish peroxidase-conjugated goat anti-rabbit, anti-mouse, and anti-goat antibodies were purchased from Invitrogen (Carlsbad, CA, USA). Actin antibody was purchased from Sigma-Aldrich (St. Louis, MO, USA).

Samples were collected in RIPA buffer (Sigma) containing Complete Protease Inhibitor Cocktail (Roche Diagnostics), and protein concentration was determined using the Bio-Rad Protein Assay. Western blots employed the following antibodies: CDK5RAP3 (sc-271776), ERK1 (sc-271270), Kras (sc-30) and c-Myc (sc-40) purchased from Santa Cruz Biotechnology; Sox2 (#2748, #3579), Oct4 (#2750), Nanog (#4893), Slug (#9585), Snail (#3879), phospho-ERK1/2 (#9101), phospho-JNK1/2 (#9251), phospho-p38 (#9211), Ras (#3965), CD44 (#3578, #3570), E-cadherin (#14472), vimentin (#3932) and cleaved caspase-3 (#9661) from Cell Signaling; N-cadherin (BD610920) and E-cadherin (BD610181) from BD Biosciences; Zeb1 (NBP-1-05987) from Novus Biologicals; and β-actin from Sigma.

Protein samples were subjected to SDS-PAGE gel electrophoresis and blotted with primary antibodies recognizing HA-tag, TAK1, β-actin, JNK1/2/3, ERK1/2, p38, NF-κB p65, phospho-JNK1/2, phospho-ERK1/2, phospho-p38, phospho-NF-κB (p65) (Cell Signaling Technology), FXRα (R&D Systems, Minneapolis, MN, USA), and HBcAg (Abcam, Cambridge, MA, USA). Protein bands were visualized using ECL Plus western blotting detection reagents (Amersham Biosciences, Buckinghamshire, UK) and an ImageQuant LAS 2000 mini system, as previously described11 (link).

The primary antibodies against the following proteins were purchased from Cell Signaling Technology (MA, USA): JNK1/2, phospho‐JNK1/2, p38, phospho‐p38, Bcl‐2, Bax, caspase 3, cleaved caspase 3 and GAPDH. The primary antibodies against ASK1 and phospho‐ASK1 were purchased from ZBTB20 Cruz Inc (TX, USA). The antibodies against tumour necrosis factor a (TNFa) were purchased from Abcam (Camb, Britain).

Protein extraction, subcellular fractionation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and immunoblot analyses were performed according to previously published procedures [33 (link)]. Briefly, the samples were separated using 7.5% gel electrophoresis and electrophoretically transferred to a nitrocellulose paper. The nitrocellulose paper was incubated with the indicated primary antibody and incubated with a horseradish peroxidase-conjugated secondary antibody. Immunoreactive proteins were visualized using electrogenerated chemiluminescence (ECL) chemiluminescence detection (Amersham Biosciences, Buckinghamshire, UK). Equal loading of proteins and the integrity of subcellular fractionation were verified using β-actin immunoblotting.
Antibodies against iNOS, p65 and IκBα were provided by Santa Cruz Biotechnology (Santa Cruz, CA, USA). Phospho-ERK1/2, ERK1/2, phospho-p38, p38, phospho-JNK1/2, JNK1/2, Lamin A/C and phospho-IκBα were obtained from Cell Signaling Technology (Danvers, MA, USA). Horseradish peroxidase-conjugated goat anti-rabbit, anti-mouse, and anti-goat antibodies were purchased from Invitrogen (Carlsbad, CA, USA). The β-actin antibody was purchased from Sigma Chemicals (St. Louis, MO, USA).

Antibody against E6AP was obtained from Santa Cruz Biotechnology (SantaCruz, CA, USA). PAI-1 antibody was purchased from BD Bioscience (San Jose, CA, USA). Phospho-JNK1/2, phospho-ERK, phospho-p38, phospho-Smad3, phospho-c-Jun, and phospho-c-Fos antibodies were procured from Cell Signaling Technology (Danvers, MA, USA). Horseradish peroxidase-conjugated goat anti-mouse and anti-rabbit antibodies were purchased from Invitrogen (Carlsbad, CA, USA). α-SMA, and β-actin antibodies, Z-Leu-Leu-Leu-al (MG132), chloroquine, and actinomycin-D were obtained from Sigma (St. Louis, MO, USA). TGF-β was provided from R&D Systems (Minneapolis, MN, USA).