Rabbit monoclonal antibody against gapdh

A total of 1 × 10⁵ cells per well of a 6-well plate were seeded. Twenty-four hours later, the cells were cultured in a medium with 1.4 ppm AgNPs in the presence or absence of α-lipoic acid or were left untreated in the control. Following incubation for 24 h, the cells were washed, and proteins were harvested by the use of a RIPA lysis buffer (both from Abcam, Cambridge, UK). Western blot analysis was performed using a semidry blotting system according to a standard protocol. Primary antibodies were rabbit polyclonal antibody against BCAT1 (Abcam, Cambridge, UK, ab197941) and rabbit monoclonal antibody against GAPDH (Cell Signaling Technology, Danvers, MA, USA, Cat# 2118, RRID: AB_561053). IRDye^® infrared dye-conjugated secondary antibodies (LI-COR Biosciences, Bad Homburg, Germany) were used. The infrared intensity was measured with an Odyssey CLx Infrared Imaging System (LI-COR).

Protein levels of AMPKα, phosphorylated AMPKα (pAMPKα), ACC, phosphorylated ACC (pACC) from fasting samples were measured by Western blot analysis as described previously [20 (link)]. Liver tissue was lysed in ice-cold lysis buffer containing 1 mmol/l dithiothreitol (DTT), 0.0025% NP40 and a cocktail of proteinase inhibitors. The lysate was centrifuged at 19,000 g for 15 min at 4°C, and the supernatant was collected as whole-cell extract. The total protein concentration of the whole-cell was measured using the Bradford reagent (Bio Rad, Hercules, CA, USA). After being heated at 100°C for 5 min, 20 μg total protein was loaded into each well, separated by 7.5% SDS-PAGE (Wako, Osaka, Japan) and transferred to a nitrocellulose membrane. The membrane was incubated with rabbit polyclonal antibodies against AMPKα, pAMPKα (Thr172), ACC, pACC (Ser79) or rabbit monoclonal antibody against GAPDH (Cell Signaling Technology, Danvers, MA, USA) at 4°C overnight. After being washed, the membrane was incubated with peroxidase-conjugated goat anti-rabbit IgG (Wako) and then visualized using an ECL system (GE Healthcare, Buckinghamshire, UK).

Cells were lysed for 30 min on ice in lysis buffer (50 mM Tris, pH 7.4, 0.15 M NaCl, 2 mM EDTA, and 1% NP-40) supplemented with Protease Inhibitor Cocktail (Roche, Mannheim, Germany). The samples were resuspended in loading buffer containing 20% beta-mercaptoethanol, boiled 5 min at 95°C, and separated by SDS-PAGE. Mouse monoclonal antibody against GFP was purchased from Roche, and rabbit monoclonal antibody against GAPDH was purchased from Cell Signaling (Frankfurt am Main, Germany). Monoclonal anti-human CYBB antibody (moAB48) was obtained from LifeSpan BioSciences (Seattle, WA, USA).

Keratocytes were cultured in 6 wells plate (5x10⁵ cells/well). After incubated with different treatments for 48 h, total protein was extracted from the lysed sample in RIPA buffer, and protein levels were quantified by protein quantitative analysis kit (Bocai, China). Samples were run on 8% SDS-PAGE gels for 2 hours at 110 V before transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, USA). After blocked in 5% bovine serum albumin in TBST for at least 1 hour, that bands were incubated with the following primary antibodies: rabbit monoclonal antibody against type VI collagen (Abcam, UK), rabbit polyclonal antibody against matrix metalloproteinase 9 (Proteintech, USA) and rabbit monoclonal antibody against GAPDH (Cell Signaling Technology, USA) in TBST overnight at 4°C. Washed 3 times with TBST, the blots were then incubated with HRP-linked secondary antibody anti-rabbit IgG (Cell Signaling Technology, USA). The results were visualized via enzyme-linked chemiluminescence by ECL kit (Thermo, USA). The densitometry of the immunoreactive bands was measured with Image J software. GAPDH was regarded as an internal control. The relative expression of Col VI and MMP9 in each group was calculated by dividing the gray value of Col VI (MMP9) to GAPDH.