Pvdf blocking reagent

The parasite lysates were treated with or without peptide N-glycosidase (PNGase) F (Roche Diagnostics, Basel, Switzerland), separated on SDS–PAGE gels, and transferred onto BioTrace PVDF membranes (PALL, Port Washington, NY). The membranes were blocked for 1 h with the protein-free PVDF Blocking Reagent (TOYOBO, Osaka, Japan). For lectin blotting, the membranes were incubated with 2 μg/mL of biotin-conjugated tomato lectin (Vector Laboratories) in 10 mM Tris HCl (pH 7.5) with 150 mM NaCl and 0.05% Tween20 (TBST); washed thrice with TBST; incubated for 30 min with Vectastain ABC reagent (Vector Laboratories); washed thrice with TBST; and developed with an enhanced chemiluminescent reagent (Promega, Fitchburg, WI), according to the manufacturer's instructions. For Western blotting, the antigen was detected with a 1:2000 dilution of polyclonal antisera in CanGetSignal reagent I (TOYOBO), followed by an HRP-conjugated anti-rabbit IgG (Promega) diluted in 1:4000 in CanGetSignal reagent II (TOYOBO).

MC3T3-E1 was treated with ALA and SFC for 1 h and then treated with LPS for 24 h. Brefeldin (3.0 μg/ml) were used to inhibit protein transport during culture. Cells were washed with PBS and treated with cell lysis buffer (5 mM EDTA, 10％ glycerol, 1％ Triton X-100, 0.1% SDS, 1％ NP-40) in PBS. Protein concentration in each of the lysates was measured with Pierce BCA Protein Assay kit (Thermo Fisher Scientific, Waltham, MA) and adjusted to be the same for each lysate. After mixing with sample buffer, it was heat denatured, were electrophoresed on a TGX Precast gel (Bio-Rad Laboratories). The proteins were transferred to a PVDF membrane, and blocked with PVDF Blocking Reagent (Toyobo Co. Ltd, Osaka, Japan). Membrane was then incubated with anti-IL6 antibody (1/2000 dilution; ProteinTech Group, Chicago, IL, USA). After washing 0.5% Tween-20 in PBS (PBS-T), the membrane was incubated with HRP-conjugated secondary antibody (Thermo Fishter Scientific, San Jose, CA). To confirm the amount of the loaded protein were equal, membrane was incubated with anti-β Actin antibody (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan). Chemiluminescence was produced using Luminata Forte (EMD Millipore Corporation, Billerica, MA) and detected with LumiCube (Liponics, Tokyo, Japan).

Equal amounts of protein were loaded onto 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels and run at 150 V for 50–60 min. Proteins were then transferred to polyvinylidene difluoride (PVDF) membranes at 100 V for 60 min. After transfer, the membranes were blocked for 1 h at room temperature in PVDF blocking reagent (TOYOBO Co. Ltd., Osaka, Japan). After three washes with Tween-Tris-buffered saline (T-TBS; 40 mM Tris-HCl, 300 mM NaCl, and 0.1% Tween 20, pH7.5), the membranes were incubated with the primary antibodies in dilution buffer overnight at 4 °C (Supplementary Table S1). After several washes in T-TBS, the membranes were incubated with anti-rabbit or mouse horseradish peroxidase (HRP)-conjugated secondary antibodies (#7074 or #7076, Cell Signalling Technology) in a dilution buffer for 1 h at room temperature. After several washes, bands were visualized using Immobilon Western Chemiluminescent HRP Substrate (Millipore Corporation, Billerica, MA, USA), and the signals were recorded using Fusion FX (Vilber, Marne-la-Vallee, France). Analyses were performed using the Evolution Capture software (Vilber). Protein phosphorylation was calculated as the ratio of phosphorylated to total protein levels and is expressed as arbitrary units. Immunodetection of β-actin was used as a loading control.

The kidney cortex was lysed with a buffer containing cell lysis buffer (Cell Signaling Technology), EDTA, and Halt Protease & Phosphatase inhibitor single-use cocktail (Thermo Fisher Scientific). Samples with an equivalent amount of protein were analyzed by SDS-PAGE. Proteins were electroblotted onto PVDF membranes (Bio-Rad laboratories) and membranes were blocked with PVDF Blocking Reagent (TOYOBO, Tokyo, Japan) then incubated sequentially with primary and secondary antibodies. Protein detection was performed using SuperSignal West Dura Extended Duration Substrate (Thermo Fisher Scientific) and LAS-4000 (Fujifilm, Tokyo, Japan).

Lysis buffer containing 50 mM HEPES, 50 mM NaCl, 50 mM NaF, 10 mM Na₄P₂O₂, 1 mM Na₃VO₄, and 5 mM EDTA, adjusted to pH 7.4, filter-sterilized, with 0.1% Triton X-100 and Complete Protease inhibitor (Roche Diagnostics, Tokyo, Japan) was used for protein extraction. Tissue samples were homogenized with 0.3 ml of lysis buffer in microtubes using a disposable pestle, centrifuged at 20,000 × g for 5 min, and the supernatant was used. After protein assay (Pearce protein assay kit, Thermo Fisher), protein concentration was adjusted to 2 mg/ml. Forty micrograms/lane was applied on a 10% precast gel (NuPAGE^® Novex gel, Thermo Fisher), and electrophoresis was performed with MOPS-SDS running buffer and transferred onto polyvinylidene difluoride (PVDF) membrane with a blotting apparatus HorizeBLOT (ATTO, Tokyo, Japan). Blotted membrane was blocked with PVDF blocking reagent (TOYOBO) for 60 min RT, staining was performed with anti-NDRG3 antibody (1:1,000, Abcam) and secondary goat anti-rabbit immunoglobulin G-horseradish peroxidase (IgG-HRP) (Santa Cruz) and HRP chemiluminescent reagent kit. For control, tubulin was visualized with anti-tubulin (1:1,000 Abcam #SC-2005) followed by goat anti-mouse IgG-HRP. Staining image was obtained using LAS3000 (Fujifilm, Tokyo, Japan). Semi-quantification from the images was performed using ImageJ (NIH) software.

Soleus muscle was homogenized (Polytron) in ice-cold RIPA buffer (25 mM Tris·HCl, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, and 0.1% SDS, pH 7.6) supplemented with Halt Protease Inhibitor Cocktail (Thermo Fisher Scientific). Homogenates were centrifuged at 14,000 g for 15 min, and the supernatant was analyzed. Protein concentration of muscle lysates was subsequently determined using a BCA Protein Assay Kit (Thermo Fisher Scientific). Muscle lysates were solubilized in Laemmli sample buffer (Bio-Rad) and heated to 100°C for 5 min. The samples (20 μg protein) were subjected to 10% SDS-PAGE, transferred to a PVDF membrane, blocked with PVDF blocking reagent (TOYOBO), and incubated with primary antibodies against stearoyl-CoA desaturase 1 (SCD1; 1:200 dilution; Santa Cruz Biotechnology) and α-tubulin (1:2,000 dilution; Abcam) overnight and, finally, with a horseradish peroxide-linked secondary anti-rabbit IgG antibody (1:5,000 dilution; Abcam). Blotted samples were analyzed using EzWestLumi plus (ATTO) and quantified by densitometry.

The extracted protein (25 μg) was separated electrophoretically in 4–15% polyacrylamide gradient precast gels (Mini-PROTEAN TGX; Bio-Rad Laboratories, Hercules, CA, USA) and transferred onto a PVDF membrane (GE Healthcare, Tokyo, Japan). The membrane was blocked with PVDF blocking reagent from Can Get Signal (Toyobo, Osaka, Japan), and subsequently incubated overnight at 4°C with anti-phosphorylated peroxisome proliferator-activated receptor γ (p-PPARγ) (Ser 112) (1:200 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA) as primary antibody. After being washed with Tris-buffered saline-Tween, the membrane was incubated for 1 h with anti-rabbit IgG secondary antibody (1:5000 dilution; GE Healthcare, Tokyo, Japan). The membranes were reprobed with anti-PPARγ (H-100) (1:200 dilution) and anti-rabbit IgG (1:5000 dilution). The bands were detected with an ECL Western Blotting Detection Reagent (GE Healthcare, Tokyo, Japan) and quantified using an Ez-Capture (ATTO, Tokyo, Japan).