Total c jun

Naringenin (95%), and chemical reagents, including Tris, NaCl, SDS and cycloheximide (CHX) were purchased from Sigma‐Aldrich (St. Louis, MO, USA). Specific antibodies against MMP‐1 were purchased from Upstate Biotechnology (Lake Placid, NY, USA). Antibodies specific to detect Ser217/221‐phosphorylated MEK, total MEK, Thr359/Ser363‐phosphorylated p90^RSK, total p90^RSK, Tyr180/182‐phosphorylated p38, total p38, Ser63‐phosphorylated c‐Jun, total c‐Jun, total c‐Fos and Ser265‐phosphrylated FRA1 were from Cell Signaling Biotechnology (Beverly, MA, USA). Antibodies specific to Thr202/Tyr204‐phosphorylated ERK1/2, total ERKs, total FRA1, alpha tubulin and lamin B were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The antibody against β‐actin was obtained from Sigma‐Aldrich. The recombinant active ERK2 protein was obtained from Upstate Biotechnology. CNBr‐Sepharose 4B, glutathione‐Sepharose 4B, [γ‐³²P] ATP and the chemiluminescence detection kit were purchased from GE Healthcare (Piscataway, NJ, USA). The protein assay kit was obtained from Bio‐Rad Laboratories (Hercules, CA, USA).

Patient-derived HNSCC tumor cells were collected and processed as previously described [9 (link), 47 (link), 48 (link)]. Retroviral vector pMSCV-Blasticidine is a gift from Dr. Mathijs Voorhoeve (Duke-NUS Medical School, Singapore). GNA13 is cloned in pMSCV-Blast vector as described earlier [27 (link)]. Stable cell lines expressing either pMSCV-Vector or GNA13 are created as described earlier [27 (link)]. ShRNAs targeting GNA13 were cloned in modified pRetro-Super vector (gift from Dr. Mathijs Voorhoeve) and stable cell lines were generated using blasticidine selection as described [27 (link)]. RPMI or DMEM complete media with 10% FBS and 1% penicillin/streptomycin (GIBCO) is used to maintain the cell lines. For Western blots, antibodies used were as follows: Gα13 (ST1629, San Diego, CA, USA), α-tubulin (010M4813, Sigma, St Louis, MO, USA), p-cJun (Thr91, ab28853, Abcam, Cambridge, UK) total cJun (9165), p-ERK1/2 (Thr202/Tyr204, 9101), Total ERK1/2 (9102), and total IκB-alpha (4812) (Cell Signaling, Boston, MA, USA) and p-IκB-alpha (Ser32, Ma5-15087, Thermoscientific, Rockford, IL, USA). Gα13 antibody used for immunohistochemistry was purchased from Sigma (HPA010087).

Livers were homogenized and protein was extracted using radioimmunoprecipitation assay buffer (RIPA, Boston BioProducts, Ashland, MA) containing phosphatase and protease inhibitors (Sigma, St. Louis, MO). Protein concentrations were normalized to 100 μg using the bicinchoninic acid (BCA) method (Pierce Chemical Co., Rockford, IL). Protein samples were separated based on molecular weight and transferred to a polyvinylidene difluoride membrane (Millipore, Billerica, MA). Membranes were incubated with primary antibodies specific for total 5′ adenosine monophosphate-activated protein kinase (AMPK), phosphorylated (Thr172) AMPK, total acetyl-coA carboxylase (ACC), phosphorylated (Ser79) ACC, total p44/42 mitogen-activated protein kinase MAPK (Erk1/2), phosphorylated (Thr202/Tyr204) Erk1/2, total c-Jun N-terminal kinase (SAPK/JNK), phosphorylated (Thr183/Tyr185) SAPK/JNK, total c-Jun, phosphorylated (Ser73) c-Jun, total p38 mitogen-activated protein kinase (P38), and phosphorylated (Thr180/Tyr182) P38 (all antibodies from Cell Signaling Technology). β-actin (Abcam, Cambridge, MA) was used as a loading control. Blots were incubated with appropriate secondary antibodies conjugated to horseradish peroxidase (HRP; GE Healthcare, United Kingdom). Western blots were repeated at least twice to ensure reproducibility.