Dc protein analysis kit

Cell lysates were prepared using a lysis buffer and the protein concentration of each sample was measured using the DC protein analysis kit (BIO-RAD Laboratories). After normalization, lysates containing the same amount of protein were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and the separated proteins were transferred onto an Immuno-Blot PVDF membrane. The membrane was blocked with 5% skimmed milk in Tris-buffered saline containing Tween-20 (TBST) for 2 h at room temperature and then incubated with the primary antibody, followed by the appropriate secondary antibody conjugated with horseradish peroxidase. Antibodies against cleaved poly ADP ribose polymerase (PARP), PD-L1, signal transducer and activator of transcription (Stat) 3, phosphorylated Stat 3, chemokine (C-X-C motif) ligand (CXCL) 8, Akt, phosphorylated Akt, ERK, and phosphorylated ERK were purchased from Cell Signaling Technology (Danvers, MA, USA). The anti-actin antibody was obtained from Santa Cruz Biotechnology. Immunoreactive bands were visualized using ImageQuant LAS 500 imager (GE Healthcare Life Sciences).

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).