Anti phospho fyn

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.