Ab8226

RIPA lysate was used to prepare tissue homogenate to extract protein and NP-40 lysate to obtain cell protein. The protein concentration was measured using the BCA protein kit (TaKaRa, Otsu, Japan, T9300A). Protein was degenerated in sodium dodecyl sulfate (SDS) buffer, followed by separating on SDS-polyacrylamide electrophoresis (PAGE) gel using electrophoresis. After that, they were transferred onto polyvinylidene difluoride (PVDF, Millipore, USA, IPVH00010) membrane and incubated with primary antibody actin (Abcam, USA, ab8226, 1 : 400), BAX (Abcam, USA, ab32503, 1 : 200), Bcl-2 (Abcam, USA, ab182858, 1 : 200), caspase-3 (Abcam, USA, ab32351, 1 : 1000), Cbfα-1 (Abcam, USA, ab113203,1 : 200), OPN (Abcam, USA, ab228748, 1 : 200), SM-α (Abcam, USA, ab7817, 1 : 500), Akt (Abcam, USA, ab8805, 1 : 200), p-Akt (Abcam, USA, ab38449, 1 : 100), ERK1/2 (Abcam, USA, ab184699,1 : 1000), p-ERK1/2 (Abcam, USA, ab278538,1 : 1000), and β-actin (Abcam, USA, ab8226, 1 : 1000) overnight at 4°C. Next, they were incubated with horseradish peroxidase- (HRP-) conjugated secondary antibodies IgG (Abcam, USA, ab150077, goat anti-rabbit, 1 : 5000) at room temperature for 45 min. After the membrane was washed in TBST, the blot was visualized by the enhanced chemiluminescence ECL kit (Thermo Fisher Scientific, NY, USA).

The tissue samples were lysed with RIPA lysate containing protease inhibitor and phosphatase inhibitor (Beyotime). The protein concentration was determined using a DC protein analysis kit (500-0111; Bio-Rad, Hercules, CA, USA) at 750 nm. The proteins were separated by sodium dodecyl sulfate polyacrylamide gels and transferred to polyvinylidene fluoride membrane. The membrane was then blocked with 5% bovine serum albumin for 1 h and probed with rabbit anti-human antibodies (1 : 1000) overnight at 4°C. Next, the membrane was reprobed with secondary antibody (1 : 5000). Luminata Western horseradish peroxidase (HRP) substrate (Millipore, Billerica, MA, USA) and Kodak XBT-1 negatives were adopted for color development. The band was quantified using Bio-Rad Quantity One software, normalized to actin level. All results were normalized with blank control values of 100%.
The antibodies used were as follows: GABA_B receptor (Invitrogen, #PA1-32248), NR2B (Abcam, Cambridge, UK; ab81271), PKC (Abcam, ab31), CaMKII (Abcam, ab171095), phosphorylation (p)-mitogen-activated protein kinase (ERK1/2) (Abcam, ab54230), p-CREB (Abcam, ab32096), and beta-actin antibody (Abcam, ab8226). Secondary antibody was mouse anti-rabbit immunoglobulin G (IgG) light chain (Abcam, ab8226).