Anti rabbit igg hrp

Fucoidan from Fucus vesiculosus was obtained from Sigma-Aldrich Co. (St. Louis, MO, USA) as fucoidan. Fucoidan from Laminaria japonica was a gift from Hi-Q Marine Biotech International, Ltd. (Taiwan) as HiQ-fucoidan. PI, NAC, anti-actin (AC-74), anti-pERK1/2 (MAPK-YT) and anti-p21 (CP74) were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Lipofectamine 2000 was purchased from Invitrogen (Grand Island, NY, USA). Anti-AKT, anti-p-AKT (S473) and anti-mouse IgG-HRP were purchased from Santa Cruz Biotechnology (CA, USA). Anti-CHOP, anti-GRP78, anti-eIF2α, and anti-rabbit IgG-HRP were purchased from GeneTex, Inc. (Hsinchu, Taiwan). Anti-caspase3 (8G10), anti-p-PERK (T980; 16F8), anti-PERK (D11A8), anti-p-eIF2α (Ser51, 119A11), anti-TLR4 (D8L5W) and anti-ATF4 (D4B8) were purchased from Cell Signaling (Beverly, MA, USA).

The extracts of primary cultures taken at 36 h or 8 days after plating were subjected to standard procedures. The protein amount was determined using BioRad protein assay. Equal amounts of protein (30 µg) were electrophoresed on 10% acrylamide gel and electrotransferred to polyvinylidene fluoride membrane, followed by immunoblotting with antibodies for myosin heavy chain (MyHC; 1:1000, GTX73432, GeneTex, Taiwan), phospho-TAK1 (1:1000, Cell signaling, #4508), p-p38 (1:1000, Cell signaling, #4511), p-JNK (1:1000, GTX52328, GeneTex, Taiwan), p-ERK (1:1000, Cell signaling, #4370), and p-NF-kB (1:1000, Cell signaling, #3033). Goat anti-actin-HRP (1:5000, Santa Cruz Biotechnology) was used for protein quantification. The membrane was washed and incubated with secondary anti-mouse IgG-HRP (1:3000, sc-2005, Santa Cruz, California, USA), anti-goat IgG-HRP (1:3000, sc-2020, Santa Cruz, California, USA), and anti-rabbit IgG-HRP (1:3000, GTX213110-01, GeneTex, Taiwan). Detection was performed using the T-Pro LumiLong Plus Chemiluminescence Detection Kit (JT96-K004M, T-Pro Biotechnology, Taiwan) and the blots were directly processed on an Amersham Imager 600 (GE, Boston, USA). Densitometry was performed using ImageJ.

The proteins were separated by 10% polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride (PVDF) membranes. After blocking in 3% BSA, the membranes were probed with primary antibodies (SREBP-1c, PPAR-α, GPAM, DGAT1, and DGAT2 (Abcam, UK); FAS, AMPK-α, and pAMPK-α (Cell Signaling Technology, Danvers, MA, CA, USA); β-actin from (Thermo Fisher Scientific, Waltham, MA, USA)) overnight at 4 °C. The membranes were washed and incubated with an anti-rabbit IgG-HRP (GeneTex, Irvine, CA, USA) for 2 h. Proteins were imaged using an advanced enhanced chemiluminescence (ECL) advanced kit (Thermo Fisher Scientific, CA, USA). Protein expression was observed using the FUSION Solo System (Vilber Lourmat, Marne-la-Vallée, France), and semi-quantified using ImageJ (NIH).