Goat anti rabbit igg conjugated to horseradish peroxidase hrp

Total protein was extracted by lysing cells in RIPA buffer containing protease inhibitor. Protein samples were separated by sodium dodecyl sulfate polyacrylamide gelelectrophoresis (SDS-PAGE) and transferred onto polyvinylidenefluoride (PVDF) membranes. After blocking with 5 % non-fat milk or 3 % BSA in TBS-T, membranes were incubated with the primary antibody. The following antibodies were used: SH2B1 (1:1000,Abcam, USA), JAK2 (1:600, Abcam, USA), p-JAK2 (1:2000, Abcam, USA), p-Rac1 (1:500, Abcam, USA), Anti-cAMP Protein Kinase Catalytic subunit (1:60000,Abcam,USA),MMP2 (1:2000,Abcam.,USA), MMP9 (1:1000, Abcam,USA), GAPDH (1:10000, Abcam, USA) and goat-anti-rabbit IgG conjugated to horseradish peroxidase (HRP) (1:5000, Santa Cruz, USA),which was used as the secondary antibody. Cells were seeded on 10 cm cell culture plates, grown to 80 % confluences, and serum starved overnight. Target signals were quantified by BandScan software (Bio-Rad, Hercules, CA) and defined as the ratio of target protein relative to GAPDH.

The chemicals used in this study were obtained from the following companies: secondary goat anti-mouse IgG conjugated to HRP (sc-2005) from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and goat anti-rabbit IgG conjugated to horseradish peroxidase (HRP), (cat. 111-035-003) from Thermo Fisher Scientific (Waltham, MA, USA). The mouse-to-mouse HRP ready-to-use kit was obtained from ScyTek Laboratories, Inc. (Logan, UT, USA). All of the other reagents and ABTS (2,20-azinobis (3-ethylbenzothiazoline-6-sulfonic acid)-diammonium salt) were purchased from Sigma (St. Louis, MO, USA) and were of the purest analytical grade. Rabbit anti-CB₁ (cat. 101500), anti-CB₂ (cat. 101550), anti-GPR55 (cat. 10224), anti-FAAH (cat. 101600), anti-MAGL (cat. 100035), and anti-NAPE-PLD (cat. 10305) polyclonal antibodies were obtained from Cayman Chemicals (Ann Arbor, MI, USA). Rabbit anti-DAGL-α (cat. PA5-23765) and anti-DAGL-β (PA5-26331) polyclonal antibodies were obtained from Invitrogen (Thermo Fisher Scientific; Waltham, MA, USA). The rabbit anti-TRPV1 (cat. TA336871) polyclonal antibody was obtained from OriGene Technologies (Rockville, MD, USA).