α actin

Vincristine, propidium iodide, anti-β-tubulin, FITC-conjugated anti-mouse IgG, poly-L-lysine hydrobromide, and SP600125 were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Suberoylanilide hydroxamic acid (SAHA) and 21-900 (HDAC/tubulin inhibitor) were synthesized by Dr. Jing-Ping Liou (School of Pharmacy, College of Pharmacy, Taipei Medical University, Taiwan). The above drugs were dissolved in DMSO (dimethylsulfoxide) and then stored at −20 °C. The acetyl-Histone H3, cdk1, caspase 3, PLK1 (p-Thr210), cyclin B1, cdk1/cdc2 (p-Tyr15), and caspase 7 antibodies were all purchased from BD Biosciences (San Jose, CA, USA). Caspase 8, PARP, MPM2 (pSer/pThr), PLK1, and α-actin were purchased from Millipore (Bedford, MA, USA). Aurora A, p-JNK, BID, BCL-2, p-BCL-2, MCL-1, BAX, BAK, Ac-α-tubulin, caspase 9, and cleaved caspase 3 were purchased from Cell Signaling Technologies (Beverly, MA, USA). The labeled secondary antibodies goat anti-mouse IgG-HRP and goat anti-rabbit IgG-HRP were purchased from Santa Cruz (Santa Cruz, CA, USA).

Western blot analysis for TNF (1:2,000, Abcam) was performed using 20 μg protein extract separated on 4–12% SDS-PAGE gels (Nupage, Invitrogen) essentially as previously described3 (link)4 (link). As a positive control, 0.5 ng 17 kDa murine recombinant TNF (Sigma Aldrich) was included. Western blotting analysis for SAPK/JNK (Cell Signaling, 1:1,000), phosphorylated (p)-SAPK/JNK (Thr183/Tyr185) (Cell Signaling, 1:1,000), p44/p42 MAPK (Cell Signaling, 1:1,000), p-p44/p-p42 MAPK (ERK1/2)(Cell Signaling, 1:1,000), p-38 (Cell Signaling, 1:1,000), p-p38 MAPK (Tyr180/Tyr182)(Cell Signaling, 1:1,000), and Iba1 (Wako, 1:500) was performed by resolving equal amounts of protein lysates by SDS-PAGE on Nupage Bis 4–12% gels, using MOPS SDS (Invitrogen) containing 0.25% antioxidant (Invitrogen) essentially as previously described6 (link)59 (link). TFIIB (1:1,000, Cell Signaling), GAPDH (1:2,500, Abcam) and α-actin (1:8,000, Millipore) were used as loading controls. SeeBlue Plus2 prestained standard (Invitrogen) was used as a molecular weight marker. Bands were quantified with Quantity One software (Biorad). Analysis was performed on 2–4 independent gels with n = 2–3/group and data were normalized to TFIIB, GAPDH or α-actin and represented as percentages relative to naïve TNF^fl/fl mice or as ratios (for MAPK).

Cells were were fixed in 4% paraformaldehyde (PFA) for 15 min, permeabilized with 0.1% Triton X-100 for 30 min, and then incubated with blocking solution (PBS + 1% BSA) at 4°C for 30 min followed by primary antibodies at 4°C overnight. Primary antibodies were diluted in Immnol Fluorence Staining Primary Antibody Dilution Solution (Beyotime, P0108) at the followings ratios: OCT4 (rabbit polyclonal antibody, 1:500, Abcam), SOX2 (rabbit polyclonal antibody, 1:500, Abcam), NANOG (rabbit polyclonal antibody, 1:500, Abcam), SSEA4 (rabbit polyclonal antibody, 1:500, Abcam), CD105 (rabbit monoclonal antibody, 1:500, Abcam), β-Ⅲ-tubulin (rabbit monoclonal antibody, 1:500, Chemicon), AFP (mouse monoclonal antibody, 1:500, Chemicon), Desmin (mouse polyclonal antibody, 1:500, Abcam), Nestin (rabbit monoclonal antibody, 1:200, Chemicon), PDX1 (rabbit monoclonal antibody, 1:500, Chemicon) and α-actin (rabbit monoclonal antibody, 1:200, Sigma). After washing three times with PBS, cells were incubated with Alexa Fluor 488 (donkey anti-mouse) or Alexa Fluor 546 (goat anti-rabbit) conjugated secondary antibody (1:500; Chemicon) for 1h at room temperature in the dark. Finally, DNA was stained with Hoechst33342 (Beyotime, C1005) for 3 min. Negative controls were processed the same way, except that the primary antibodies were replaced with blocking buffer.

Muscle SREBP-1c, ACLY, LXRs, FAS, MAFbx, MuRF-1, MyoD, Myostatin, IL-1β, TNF-α, and NCAM levels were determined using the Western blot technique [27 (link)]. The muscle homogenates were prepared in ice-cold lysis buffer. SDS-PAGE sample buffer containing 2% β-mercaptoethanol was added to the supernatant. Twenty micrograms of protein were electrophoresed and then transferred into Nitrocellulose membranes (Schleicher and Schuell Inc., Keene, NH, USA). Nitrocellulose blots blocked with 1% bovine serum albumin in PBS for one hour prior to administration of the primary antibodies (SREBP-1c, ACLY, LXRs, FAS, MAFbx, MuRF-1, MyoD, Myostatin, TNF-α, IL-1β, and NCAM) (Abcam, Cambridge, UK) that were diluted (1:1000) in the same buffer containing 0.05% Tween-20. Protein loading was checked using an antibody against α-actin (A5316; Sigma Aldrich, St. Louis, MO, USA). Bands were analyzed densitometrically using an image analysis system (Image J; National Institute of Health, Bethesda, MA, USA).

Briefly, paraffin-embedded sections (10-μm thick) were deparaffinized, treated with 3% H₂O₂ in methanol for 10 min to inactivate any endogenous peroxidase, washed with PBST (0.1% Tween 20 in PBS), incubated for 60 min in blocking buffer (3% bovine serum albumin [BSA] in PBST), incubated for 60 min with primary antibodies to inducible nitric oxide synthase (iNOS), Ras (Santa Cruz Biotechnology), α-actin (Sigma, St. Louis, MO, USA), proliferating cell nuclear antigen (PCNA) (Dako Cytomation, Carpinteria, CA, USA), IL-6, TNF-α, β-galactosidase, (Abcam PLC, Cambridge, UK), and adenosine 5′-monophosphate-activated protein kinase (p-AMPK; Cell Signaling, Beverly, MA, USA), all diluted in 1% BSA, washed three times for 10 min in PBST, incubated 60 min with the horseradish peroxidase (HRP)-conjugated secondary antibody (Sigma) diluted in 1% BSA at 37 °C, and finally incubated 3 min at room temperature with 3, 3′-diaminobenzidine (DAB) for color development. All experiments were repeated three times.