Table 1. Proteins differentially regulated by chronic Δ9-THC in intact and ovarectomized rats.

DeCyder software (GE Healthcare) was used for simultaneous comparison of abundance changes across all samples’ 2-DE gels, and for comparisons of individual Cy3 and Cy5 samples for each rat. The DeCyder differential in-gel analysis (DIA) module was used for pair-wise comparisons of each sample on a gel to the Cy2 labeled standard present on each gel. For each pair-wise DIA comparison, the entire signals from each CyDye channel are normalized prior to the co-detection of protein spot boundaries and the calculation of the volume ratio for each protein spot. The DeCyder biological variation analysis (BVA) module was then used to simultaneously match all protein spot maps from all gels, and using the Cy3/Cy5:Cy2 DIA ratios, calculate abundance changes and paired Student’s t-test p-values for the variance of these ratios for each protein pair across all samples. In the absence of sufficient experimental replicates, no statistical information can be generated. Fold abundance changes are reported, whereby a fold increase is calculated directly from the volume ratio, and a fold decrease by the inverse of volume ratio. The DIA module was also used to calculate the direct Cy5:Cy3 volume ratio for each paired sample individually (without the Cy2 mixed standard) to assess the contribution from each subject and reveal changes that were present in a group. After determining spots of interest, the protein were excised using the Ettan Spot Handling Workstation with a 2-mm diameter spot-picking head (GE Healthcare). Gel spots were cut, de-stained and then digested with trypsin (Promega). The resulting peptide mixture was loaded on a Dionex PepMap C18 trap column and was separated by a New Objective reversed phase C18 Picofrit column/emitter. Peptide mass was determined by a Thermo-Fisher LTQXL linear ion trap mass spectrometer (Waltham, MA, USA) coupled with an Eksigent nanoLC (Dublin, CA, USA). The raw data were analyzed by the Mascot search engine V2.2 (Matrix Science Inc, Boston, MA, USA) against the rat SwissProt database (false discovery rate <5%) to generate a list of possible proteins for that gel spot.

Matched Protein1	Accession1	Protein Score	Peptides Matched	Coverage (%)	Differences (expressed as a ratio to intact/saline grup)			1-ANOVA	Subcellular Localization1
					Intact/Δ9-THC	OVX/saline	OVX/Δ9-THC
pyruvate carboxylase	P52873 (PYC_RAT)	254	36	43	0.79	0.60	0.46	0.0075	mitochondria
NADH dehydrogenase	Q561S0 (NDUAA_RAT)	595	19	47	0.87	0.56	2.51	0.4100	mitochondria,
NM23 (NDKB)	P19804 (NDKB_RAT)	165	9	57	1.70	2.24	3.31	0.0480	cytosol, plasma membrane
translationally controlled tumor protein (TCTP)	P63029 (TCTP_RAT)	90	6	39	0.62	2.29	2.82	0.0330	cytosol
DJ-1 (PARK-7)	O88767 (PARK7_RAT)	127	16	73	1.78	2.14	2.24	0.0240	cytosol, nucleus
AHA1	B0BN63 (AHSA1_RAT)	298	12	49	1.26	2.09	1.55	0.0430	cytosol, ER

¹- Matched Protein, Accession and Subcellular Localization information as listed in The Universal Protein Resource (The Uniprot Consortium, 2010)