Sv5 pk1

Western blotting was performed using the following antibodies: peroxidase-anti-peroxidase (PAP) (P1291, Sigma, 1:2000); anti-Calmodulin binding protein (CBP) (RCBP-45A-Z, ICLab, 1:500); anti-tubulin (B-5–1–2, Sigma, 1:5000); anti-FLAG (M2, F1804, Sigma, 1:1000); anti-MYC (9E10, Agro-Bio LC; 9E11, ab56, Abcam, 1:1000; or rabbit polyclonal ab9106, Abcam, 1:1000); anti-V5 (SV5-Pk1, AbD Serotec, 1:1000); anti-HA (16B12, Ozyme; rabbit polyclonal, ab9110, Abcam, 1:1000). Protein concentrations were measured by the Bradford method and used to load equal amounts of proteins across samples. Quantification of signal intensity was performed using staining, film exposure or digital acquisition that were within the linear range of detection, as verified by loading serial dilutions of one sample, and analysed using ImageJ.

For Igo1-myc8, 10 μg whole-cell extracts were run on 12% acryl–bisacrylamide (29:1) gels and 8.5% acryl–bisacrylamide (29:1) 25 µM Phos-tag gels (Wako Chemicals GmbH, Germany). For Whi5-PK3, 10 μg whole-cell extracts were run on 12.5% acryl–bisacrylamide (125:1) gels and 8.5% acryl–bisacrylamide (29:1) 5 µM Phos-tag gels. Proteins were transferred to nitrocellulose membranes (Protran; Amersham, GE Healthcare, Germany) using wet blotting and revealed using standard immuno-blotting and ECL procedures. Western blot quantifications were performed from images acquired with the multi-application gel imaging system PXi 4 (Syngene, A Division of Synoptics Ltd, UK), using ImageJ. Primary antibodies were mouse monoclonal anti-myc (1:1000 from ascites for Igo1-myc8), rabbit polyclonal anti-swi6 (1:100,000) and mouse monoclonal anti-PK (1:5,000; clone SV5-Pk1; Bio-Rad Antibodies, France).

HEK293FT cells were seeded at a density of ~7 × 10⁵ cells/well in a 6-well plate with DMEM containing 2% FBS. After overnight culture, cells were transfected with pcDNA3.2-GPR55-V5 using PEI MAX and Opti-MEM for 24 h, followed by incubation with curcumin for 3 h. The cells were washed with PBS(−) and sonicated in lysis buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2 mM EDTA, 1% TritonX-100, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM sodium orthovanadate, 10 μg/mL leupeptin, 1 μg/mL aprotinin, 1 mM phenylmethylsulfonyl fluoride). After centrifugation (20,000 × g for 10 min), the supernatant was analyzed by western blotting with monoclonal anti-V5 (SV5-Pk1, Bio-Rad, Hercules, CA, USA, RRID:AB_322378) or monoclonal anti-β-actin antibody (2D4H5, Proteintech, Rosemont, IL, USA, RRID:AB_2687938). After reaction to horseradish peroxidase-conjugated secondary antibody (Bio-Rad, RRID:AB_11125547), the immunoreactive bands were developed with Immobilon Western Chemiluminescent HRP substrate (Millipore, Bedford, MA, USA) and were detected with LAS4000 imager (GE Healthcare, Piscataway, NJ, USA). Band intensities were determined using ImageJ software version 1.53k (NIH, Bethesda, MD, USA). All blots were processed in parallel and derived from the same experiment.