Tissue from cultures was harvested for cell fractionation either immediately following treatment or after a 4 day withdrawal. Prior to scraping, cultures were washed three times to remove excess media in an ice-cold HEPES-buffered sucrose solution containing (in mM): 200 sucrose, 1.9 KCl, 6 MgCl2, 0.5 CaCl2, 10 glucose, 0.4 ascorbic acid, 25 HEPES, pH 7.3 with KOH.
A crude membrane fraction was prepared and tissue samples were used for western blotting as previously described (Mulholland and Chandler, 2010 (link)). After separation on NuPage Novex gels (4–12% Bis-Tris; Invitrogen Corp., Carlsbad, CA) protein was transferred to Immobilon-P PVDF membranes (Millipore Corporation, Billerica, MA). Membranes were blocked in 1% non-fat dry milk plus 5% BSA and incubated at 4°C overnight in a 1:500 dilution of the L15 anti-CB1 primary antibody (kindly provided by Dr. Ken Mackie, Indiana University, Bloomington, IN). A horseradish peroxidase (HRP) conjugated goat anti-rabbit secondary antibody was then added and bands were detected using enhanced chemiluminescence (ECL). The band corresponding to the molecular weight of CB1 (≈52 kDa) was quantified using computer-assisted densitometry with ImageJ v1.41 (National Institutes of Health, USA).
A crude membrane fraction was prepared and tissue samples were used for western blotting as previously described (Mulholland and Chandler, 2010 (link)). After separation on NuPage Novex gels (4–12% Bis-Tris; Invitrogen Corp., Carlsbad, CA) protein was transferred to Immobilon-P PVDF membranes (Millipore Corporation, Billerica, MA). Membranes were blocked in 1% non-fat dry milk plus 5% BSA and incubated at 4°C overnight in a 1:500 dilution of the L15 anti-CB1 primary antibody (kindly provided by Dr. Ken Mackie, Indiana University, Bloomington, IN). A horseradish peroxidase (HRP) conjugated goat anti-rabbit secondary antibody was then added and bands were detected using enhanced chemiluminescence (ECL). The band corresponding to the molecular weight of CB1 (≈52 kDa) was quantified using computer-assisted densitometry with ImageJ v1.41 (National Institutes of Health, USA).
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