. Author manuscript; available in PMC: 2014 Oct 1.

Published in final edited form as: Biochim Biophys Acta. 2013 Jun 15;1834(10):2045–2056. doi: 10.1016/j.bbapap.2013.06.003

Table 1. Expression levels and functional activity of CB₂ in Rho-tag fusion constructs.

To quantify the relative levels of the constructs expressing Rho-tagged CB₂ with respect to the control (CB₂-130), the analysis of Western blot probed with anti- CB₂ antibody (Fig. 1A) was performed by densitometry using the Kodak 1D program for data analysis. The intensity corresponding to CB₂-130 was arbitrary chosen as 100%. The activity values for each construct presented in Fig. 1 B were calculated as a percentage of the activity displayed by the control CB₂-130. For calculation of specific activity (%), the activity values (CPM) were divided by the amount of CB₂ present in the corresponding band, normalized to 1 μg of total protein in membranes, and calculated as a percentage of the specific activity of CB₂-130. The expression level was estimated to be 3 ng of CB₂-130 per μg of total protein [2]. For qualitative assessment of Rho-tag accessibility in the fusion CB₂, intensity of bands from Western blots visualized with 1D4 (Fig. 1 C) were normalized by dividing them by intensities of bands visualized with anti-CB₂ antibody (Fig. 1 A). Constructs with ratios of 2-3 were classified as having highly accessible tag (+++); ratios of 1-2, medium accessible tag (++), ratios of 0.5-1, low accessible tag (+) and ratio <0.5 as not accessible (−).

Construct	Fusion protein components	CB₂ %	Activity % (G-protein activation)	SA⁴ %G-protein activation)	Accessibility of lD4	B_max (pmo1 CP 55940/mg total protein)	K_D (nM)
130 (control)	MBP-TEV-Strep-CB₂-His₁₀	100	100	100	NA	2.4 ± 0.2	2.2 ± 0.4
125 (control)	MBP-TEV-Strep-CB₂-TEV-TrxA-His₁₀	123	80.4	65	NA	ND	ND
181	MBP-TEV-Step-*Rho*-CB₂-His₁₀	71	33.4	47	−	0.4 00.4	2.2 ± 0.5
182	MBP-TEV-CB₂-*Rho*	30	9.6	32	++	ambiguous	ambiguous
183	MBP-TEV-Strep-CB₂-*Rho*-His₁₀	177	68.4	39	−	1.9 ± 0.3	3.0 ± 0.8
250	MBP-TEV-CB₂-His₁₀-*Rho*	43	6.2	15	+	ND	ND
251	MBP-TEV-His₁₀-CB₂-*Rho*	18	17.9	98	++	ND	ND
252	MBP-TEV-His₁₀-CB₂-TEV-TrxA-*Rho*	120	96.1	80	+++	2.8 ± 0.7	2.0 ± 1.2
253	MBP-TEV-CB₂-spacer 1-*Rho*¹	53	21.0	40	+++	ND	ND
254	MBP-TEV-CB₂-spacer 2-*Rho*²	55	76.1	138	+++	ND	ND
255	MBP-TEV-His₁₀-CB₂-spacer 3-TrxA-*Rho*³	98	94.0	96	+++	7.8 ± 4.2	6.2 ± 4.6

Spacer 1: A₂HMG₃SAS

Spacer 2: A₂HMA₃N₅AS

Spacer 3: A₃N₅G₃SENLYG₂SG₃SEF

⁴

Specific activity

Table 1. Expression levels and functional activity of CB2 in Rho-tag fusion constructs.

Table 1. Expression levels and functional activity of CB₂ in Rho-tag fusion constructs.