Gel logic imaging system

The total protein fraction was extracted using M-PER buffer (Thermo Fisher Scientific, USA) with protease and phosphatase inhibitors (Sigma Aldrich, USA). The protein concentration was determined by Bradford method. After SDS-PAGE and proteins transferred on PVDF membranes were incubated overnight at 4 °C with primary antibodies (Table 2), and subsequently detected with HRP-conjugated goat anti-rabbit IgG secondary antibody (1:4000; Santa Cruz Biotechnology, USA). The membranes were developed with SuperSignal West Pico Chemiluminescence Substrate (Thermo Fisher Scientific, USA) with Gel Logic Imaging System (Kodak, USA). Pictures of full uncropped membranes can be found in ‘Supplementary materials uncropped WBs’.Table 2

Antibodies utilized for protein detection.

Antibody target	Manufacturer	Cat no
AMPKα	Cell Signaling	#2793
Phospho-AMPKα	Cell Signaling	#4188
AMPKβ1	Cell Signaling	#4178
Phospo-AMPKβ1	Cell Signaling	#4186
GFP	Cell Signaling	#2955
GAPDH	Cell Signaling	#2118
goat anti-rabbit IgG-HRP	Santa Cruz Biotechnology	sc-2004
goat anti-mouse IgG-HRP	Santa Cruz Biotechnology	sc-2005
LKB1	Santa Cruz Biotechnology	sc-32245
Phospho-LKB1	Cell Signaling	#3482
Tuberin	Cell Signaling	#4308
Phospho-Tuberin	Cell Signaling	#23402
NFκB	Cell Signaling	#8242
SNAIL	Cell Signaling	#3895

The recombinant CB₂ protein was detected by Western blot with mouse monoclonal antibody against human cannabinoid receptor CB₂ (R&D Systems) or monoclonal anti-His6 antibody (Qiagen). The Western blot was developed with anti-mouse HRP antibody (1:5000 dilution) and visualized by chemiluminescence with Gel Logic imaging system (Kodak). The concentration of the detergent-solubilized protein was determined with a UV–Vis spectrometer (Agilent Technologies) using DC Protein Assay Reagent (BioRad), and bovine serum albumin (BSA) as protein standard.

Total cellular RNA was isolated using the RNeasy kit (Qiagen). For standard reverse transcriptase-polymerase chain reaction (RT-PCR), 400 ng RNA was reverse transcribed using random hexamers and SuperScript II Reverse Transcriptase (Life Technologies) followed by PCR with Platinum Taq PCR Polymerase (Life Technologies) according to the manufacturer's instructions. PCR product was resolved on 5% non-denaturing polyacrylamide gel and stained in ethidium bromide prior to imaging on the Kodak Gel Logic Imaging System. The percentage of exon skipping was calculated as [exon exclusion band/ (exon inclusion band + exon exclusion band)] x 100 with a correction factor for the amount of ethidium bromide per base pair of DNA. See Supplementary Table S3 for primer sequences.
For quantitative RT-PCR, approximately 10 ng RNA was added to EXPRESS One-Step SuperScript qRT-PCR Universal (Life Technologies) with custom designed or predesigned Taqman primer and probe sets (see Supplementary Table S4 for sequences). All quantification was performed by the relative standard curve method and normalized to Ribogreen.