Anti gpr55

Chemicals were of the purest analytical grade. MEM, glutamine, PES, DMSO, collagenase, DNase1, trypsin, BPA (Bisphenol A), ABTS (2,2′-azinobis [3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt), and hyaluronidase were purchased from Sigma (St. Louis, MO, USA). Rabbit anti-CB₁, anti-CB₂, anti-GPR55, anti-FAAH, anti-DAGL-α, anti-DAGL-β, anti-MAGL, anti-NAPE-PLD polyclonal antibodies, and KT109 were purchased from Cayman Chemicals (Ann Arbor, MI, USA). Mouse anti-actin antibody was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA), and rabbit anti-TRPV1 polyclonal antibody was from OriGene Technologies (Rockville, MD, USA). Rabbit anti-inhibin B polyclonal antibody was obtained from Elabscience Biotechnology (Houston, TX, USA), and rabbit anti-transferrin polyclonal antibody was from Proteintech Group (Chicago, IL, USA). Iodoresinferatoxin was obtained from Tocris Cookson (Bristol, UK), whereas SR144528 was a kind gift from Sanofi-Synthelabo (Montpellier, France).

Control THP-1 macrophages or THP-1-derived foam cells, seeded at 3x10⁶ cells/well, were washed 3 times with cold phosphate buffer saline (PBS), incubated for 3 min with accutase and collected for cell lysis. Then, cells were lysed with RIPA buffer, and protein expression assessed by Western blotting, as reported [30 (link)]. Cell homogenates were subjected to 10% SDS-PAGE (40 μg/lane) under reducing conditions, then gels were electroblotted onto 0.45-mm nitrocellulose filters and were immunoreacted with anti-GPR55 (1:200, Cayman), anti-ABCA1 (1:1000, Abcam), anti-ABCG1 (1:1000, Abcam), anti-SRBI (1:1000, Abcam) or with anti-β-actin monoclonal antibody (1:20000, Sigma Aldrich). Proteins were detected by enhanced chemiluminescence (ECL; Amersham Pharmacia Biotech) and by exposure to X-ray film (Hyper ECL; Amersham Pharmacia Biotech), quantitating band intensity by densitometry in a ChemiImager 4400 apparatus (Alpha Innotech, San Leandro, CA).

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).