Western lightning plus ecl

After treatment and at the indicated time, cells were washed once with PBS and lysed with 6 M urea buffer equipped with protease and phosphatase inhibitors (PMSF, aprotinin, pepstatin, leupeptin, sodium orthovanadate, and sodium pyrophosphate). Protein concentration was measured using Bradford reagent (Sigma-Aldrich, Germany). Equal amounts of protein (50 μg) were loaded into each well of 10% SDS-PAGE for resolving the samples. Subsequently, separated proteins were blotted onto a PVDF membrane (GE Healthcare, Germany). Membranes were then washed with TBS/tween and blocked for 3 h at room temperature with fat-free dried milk before incubation with the indicated primary antibodies at 4 °C for overnight.
Anti-β-actin and anti-vinculin antibodies were purchased from Santa Cruz Biotechnology, whereas anti-mouse and rabbit IgG horseradish peroxidase (HRP)-linked antibodies were purchased from Cell Signaling Technology.
Membranes were developed using Western Lightning^TM Plus-ECl (Perkin Elmer, Germany) as HRP substrate. Proteins of interest were detected using a Fujifilm LAS-3000 imaging system.

After treatment, cells were harvested, washed once with PBS, and lysed using 6 M urea buffer supplemented with a cocktail of protease and phosphatase inhibitors, namely aprotinin, leupeptin, pepstatin, PMSF, sodium orthovanadate, and sodium pyrophosphate. Protein content of samples was determined using Bradford reagent (Sigma-Aldrich). SDS-PAGE was subsequently performed to resolve the samples, and proteins were then transferred onto PVDF membranes (GE Healthcare, Munich, Germany). Membranes were washed once in TBS-Tween for 5 min, and then blocked for 1 h at room temperature using 5% non-fat dry milk in TBS/Tween. Membranes were then washed once in TBS/Tween for 5 min and were incubated overnight at 4 °C with the primary antibody. Anti-VDUP1 (TXNIP) antibody was purchased from MBL, whereas anti-β-actin and anti-vinculin antibodies were purchased from Santa Cruz Biotechnology. Anti-ITCH, anti-phospho-ACC ^(S79), as well as anti-mouse and rabbit IgG horseradish peroxidase (HRP)-linked antibodies were purchased from Cell signaling technologies. Western Lightning^TM Plus-ECl (Perkin Elmer) was utilized as HRP substrate. Target proteins were detected using the Fujifilm LAS-3000 imaging system.

The process of Western blotting was followed the protocol in literature^{37 (link)}. The blots were pulsed with Western Lightning^TM Plus-ECL (Perkin-Elmer Life Sciences) and the signals were determined by the intensity of chemiluminescence by LAS-3000 imager (Fujifilm, Tokyo, Japan).

The rTFF1 produced by E. coli BL21(DE3) (pET-TFF1) and B. choshinensis (pNCMO2-TFF1) at various time expression intervals were analyzed by Tricine-SDS-PAGE [37 (link)]. The yield of rTFF1 was estimated by comparing the density of the rTFF1 bands to standards (serial dilutions of 103 mg/L purified rTFF1) on SDS-PAGE gels analyzed using Gel-Pro Analyzer^TM version 3.0 (Total-Integra Technology Co., Ltd, Taipei, Taiwan). The electrophoresis gel was then transferred into PVDF membranes (Millipore, Darmstadt, Germany). The mouse anti- His•tag (Millipore, Darmstadt, Germany) and goat anti-mouse HRP (Millipore, Darmstadt, Germany) were used to immunize the rTFF1. The presentations of rTFF1s were then stained with Western Lightning^TM Plus-ECL (Perkin Elmer, Inc., USA), analyzed by Fusion-Capt advance analysis software (Vilber Lourmat, France).

Embryos were injected 4x at the four-cell stage and animal caps were prepared at stage 9. At stage 28, 15 caps per condition were pooled in 100 µl TNMEN-150 lysis buffer (150 mM NaCl, 1 mM EDTA, 2 mM MgCl2, 0.1% Nonidet-P40, 50 mM This pH8.0, 1x Roche cOmplete (#04693116001)). Co-Immunoprecipitation was performed following standard protocol, using 10 µl magnetic beads (Dynabeads M-280 Sheep Anti-Mouse IgG; #11202D) and 0.4 µl monoclonal mouse anti-FLAG antibody (Sigma; #F3165) per sample for 2 hr at 4°C. 10% of sample was removed prior to treatment with antibody/magnetic beads (input), and 30 µl of sample was removed after the treatment (supernatant). SDS-Page and Western blotting were performed using standard procedures using a 10% separating gel, Milipore Immobilon-FL PVDF membrane (#IPFL00010), TBS containing 0.1% Tween-20 (TBSw) for washing, TBSw plus 5% non-fat dry milk for blocking. FLAG-/GFP-tagged proteins were detected using monoclonal mouse anti-FLAG antibody (1:2000, Sigma; #F3165), polyclonal rabbit anti-GFP antibody (1:2000, Abcam; #ab290), anti-rabbit/-mouse HRP conjugated secondary antibodies (1:5000, Bio-Rad; Goat anti-Rabbit IgG #1706515 and Goat anti-Mouse IgG #1706516), Western Lightning Plus-ECL (Perkin Elmer; #NEL103E001EA), and Amersham Hyperfilm ECL (GE Healthcare Life Sciences; #28906836).

Cells were harvested at 60–70% of confluency in cold PBS and lysed with Laemmli buffer (2x concentrated) supplement by 2 mM PMSF, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 5 mM EGTA, 1 mM EDTA, 2 mM Na₄P₂O₇, 5 mM NaF and 5 mM Na₃VO₄. Samples containing equal amounts of protein per lane were loaded, resolved in SDS–PAGE and then transferred onto nitrocellulose membrane. The membranes were incubated for 1 h in 5% skimmed milk and probed overnight with specific primary antibodies at 4°C. Secondary antibodies conjugated with HRP (Sigma-Aldrich) and Western Lightning Plus-ECL (PerkinElmer) were used to visualize specific proteins. For immunoprecipitation experiments, cells were lysed in lysis buffer containing 1% Triton X-100. Supernatant was incubated with 2 μg of appropriate antibody overnight at 4°C. The samples were incubated with protein A or protein G beads (Santa Cruz Biotechnology, sc-2001, sc-2002) according to the manufacturer's instructions. Immunocomplexes were eluted from beads with Laemmli buffer and analysed by western blotting.