Anti mouse and rabbit igg horseradish peroxidase hrp linked antibodies

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.