Anti phospho plcγ1

The cells were scraped from the plates using RIPA Lysis Buffer (Merck Millipore, Darmstadt, Germany) containing a protease and phosphatase inhibitor cocktail (Roche, Basel, Switzerland). After incubation on ice for 30 min, the cell lysates were centrifuged at 14,000 rpm for 20 min at 4°C. Proteins were quantified using BCA protein assay (Thermo Scientific, Waltham, MA, USA). The protein lysates were resolved on 10% sodium dodecyl sulfate polyacrylamide gels and then transferred to a polyvinylidene difluoride membrane. Anti-COX-2, anti-phospho-cPLA₂, anti-phospho-Lyn, anti-phospho-Syk, anti-phospho-Fyn, anti-phospho-PLCγ1, anti-phospho-ERK, anti-phospho-Akt, and anti-α-tubulin antibodies (Cell Signaling Technology, Boston, MA, USA) were used to detect COX-2, p-cPLA₂, p-Lyn, p-Syk, p-Fyn, p-PLCγ1, p-ERK, p-Akt, and α-tubulin, respectively. α-Tubulin was used as protein loading control. Blots were observed using a western blot detection kit (Thermo Scientific, Waltham, MA, USA). Protein bands were quantified using Image Lab software (Bio-Rad Laboratories, Richmond, CA, USA).

The cells were scraped from the plates with RIPA lysis buffer containing a protease and phosphatase-inhibitor cocktail (Roche, Basel, Switzerland). The lysates were quantified using the BCA protein assay kits (Thermo, MA, USA). Equal amounts of protein were resolved on 10% SDS-PAGE and then transferred to a PVDF membrane. Anti-COX-2, anti-β-actin, anti- phospho-Lyn, anti-phospho-Syk, anti-phospho-Fyn, anti-phospho-PLCγ1, anti-phospho-cPLA2, anti-phospho-ERK, and anti-phospho-Akt (Cell Signaling, MA, USA) were used to detect COX-2, β-actin and the phosphorylated form of Lyn, Syk, Fyn, PLCγ1, cPLA2, ERK, and Akt, respectively.

Ginseng Radix, Pinellia Tuber, Bupleurum Root, Glycyrrhizae Radix et Rhizoma, Scutellaria Root, Zingiberis Rhizoma Crudus and Zizyphi Fructus were purchased from Yeongcheon herbal market (Yeongcheon, Korea). Identification of all plant material was confirmed by Prof. Ki Hwan Bae of the College of Pharmacy, Chungnam National University (Daejeon, Korea), and all voucher specimens were deposited in the herbal bank in Korea Institute of Oriental Medicine (KIOM, Daejeon, Korea). Dulbecco’s Modified Eagle Medium (DMEM) was purchased from Lonza (Wakersville, MD, USA). Fetal bovine serum (FBS) and phosphate-buffered saline (PBS) were purchased from Hyclone (Longan, UT, USA). Penicillin/streptomycin and trypsin/EDTA were purchased from Gibco (Grand Island, NY, USA). Anti-phospho-ERK1/2, anti-phospho-Akt, anti-phospho-PLCγ1, anti-ERK1/2, anti-Akt, anti-PLCγ1, anti-CDK2, anti-CDK4, anti-cyclin D₁, anti-cyclin E₁ and anti-β-actin antibodies were from Cell Signaling Technology Inc. (Beverly, MA). Anti- phospho-proliferating cell nuclear antigen (PCNA) was purchased from Abfrontier (Seoul, Korea). PDGF-BB was obtained from Upstate Biotechnology (Lake Placid, NY, USA). Cell Counting Kit-8 (CCK-8) was purchased from Dojindo Molecular Technologies (Rockville, MD, USA). Other chemicals were of analytical grade.