Mammalian protein lysis buffer

Western blot was performed according to previous studies [24 (link), 32 (link)] to assess inflammation (COX-2) and collagen deposition (α-SMA, MMP-9) to further delineate the mechanism of emodin (SMAD3). Mammalian protein lysis buffer (Thermo Fisher Scientific, Waltham, MA, USA) was used to extract the total tissue protein. The same amounts of protein were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to polyvinylidene fluoride membranes (EMD Millipore, Billerica, MA, USA). The membranes were incubated in the diluted primary antibodies overnight and stored at 4°C. The primary antibodies were anti-COX-2 antibody (Santa Cruz, 1 : 200 dilution), anti-SMAD3 antibody (Santa Cruz, 1 : 500 dilution), anti-α-SMA antibody (Santa Cruz, 1 : 200 dilution), and anti-beta-actin (β-actin) antibody (Santa Cruz, 1 : 1000 dilution). The membranes were incubated with the secondary antibody followed by horseradish peroxidase (HRP; Santa Cruz). Then, an enhanced chemiluminescence system (EMD Millipore) was used to detect the bands. The intensity of the bands was calculated using Image-Pro Plus 5.0 software (Media Cybernetics Inc., Rockville, MD, USA).

Western blot analysis was performed according to previously published methods [23 (link)]. The cell samples were briefly lysed with mammalian protein lysis buffer (Thermo Fisher Scientific) containing a protease inhibitor mixture (Roche Applied Science, Switzerland). Twenty micrograms of total protein was separated by 10% (wt/vol) SDS-PAGE and transferred onto a nylon membrane (Merck Millipore, Germany). After being blocked with 2% (wt/vol) bovine serum albumin (Sigma-Aldrich, Saint Louis, MO 63103, USA), the antibodies were then used to probe the membrane. The following antibodies were used: histone H2A (CST, 12349, Danvers, Massachusetts, USA) 1 : 1000, LDLR (Millipore, MABS26) 1 : 1000, PCNA (CST, 13110) 1 : 1000, PCSK9 (Circulex, CY-P1037, MBL, Japan) 1 : 1000-2000, β-actin (CST, 4970) 1 : 1000, GAPDH (CST, 5174) 1 : 1000, Akt (pan) (CST, 4691) 1 : 1000, phospho-Akt (Ser473) (CST, 4060) 1 : 1000, p44/42 MAPK (Erk1/2) (CST, 4695) 1 : 1000, and phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (CST, 4370) 1 : 1000. The secondary antibodies used in the western blotting are as follows: anti-mouse IgG, HRP (CST, 7076) and anti-rabbit IgG, HRP (CST, 7074). The protein expression in the western blot analysis was measured by gray analysis software (ImageJ).