Each rhodopsin molecule binds its own arrestin

Hanson et al. 10.1073/pnas.0610886104.

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SI Methods
SI Figure 3




Fig. 3. Quantification of arrestin and rhodopsin by Western blot. (A and C) Representative Western blots for rhodopsin (A) and arrestin (C) quantification. Because rhodopsin always appears as a series of bands believed to represent mono- and oligomers, the sum of the signal in all rhodopsin bands (indicated by arrowheads in A) in each lane was used for quantification. (B and D) Standards (filled circles) used to construct calibration curves (lines) and quantification of rhodopsin (B) and arrestin (D) in experimental samples (open circles) in lanes A-F (A and B) and A-L (C and D), respectively. Note that lane D in C represents a retinal sample from arrestin knockout mouse.





SI Methods

Tissue Preparation, Immunohistochemistry, and Quantitative Image Analysis. The front half of the enucleated eye was cut off with a razor blade to remove the lens and cornea, and the eyecup was immediately immersed into fixing solution (4% paraformaldehyde in 0.1 M cacodylate buffer, pH 7.2) and incubated for 4 h at room temperature in the dark (dark-adapted eyes) or in room light (light-adapted eyes). The eyecups were rinsed three times with 0.1 M cacodylate buffer, pH 7.2, cryoprotected overnight in 30% sucrose, and then frozen at -80°C. Fixed eyecups were cut into 30-mm-thick sections. Free-floating sections were blocked for 1 h in PBS with 0.3% Triton X-100 and 3% BSA at room temperature and then incubated with rabbit polyclonal anti-arrestin antibody (1:500; a generous gift of Dr. Narsing A. Rao, University of Southern California) in PBS with 0.03% Triton X-100, 1% BSA (IC buffer). We found that the most quantitative staining is achieved after 48 h of incubation of free-floating sections at 4°C, as judged by the intensity of staining of WT and arrestin hemizygous (Arr+/-) mice. Retinas from arrestin knockout (Arr-/-) mice were used as a control to gauge the signal-to-noise ratio. After washing with IC buffer, the sections were sequentially incubated with chicken anti-rabbit-Alexa 488 and goat anti-chicken-Alexa488 antibodies (Molecular Probes) at a dilution of 1:1,000 for 1 h each at room temperature. After the final wash with IC buffer, the sections were mounted on a coverslip by using Antifade reagent (Molecular Probes) and viewed by using the 40 ´ 1.3 PlanNeoFluar objective lens of a Zeiss LSM 510 laser scanning confocal microscope. The immunofluorescence was detected by excitation with a 488-nm laser line and an LP505 filter. The same settings for the acquisition of all images were used to enable subsequent quantitative analysis. DIC images were acquired in parallel to facilitate the identification of subcellular compartments. The total amount of fluorescence in the outer segments, inner segments, and outer nuclear layer and synaptic terminals, and background in the extracellular regions was determined by using MetaMorph software by multiplying the area of each compartment by the average fluorescence intensity in that compartment minus average intensity of background fluorescence. The percentage of arrestin in the outer segments was calculated based on these measurements (shown in Table 1).

Statistical Analysis. The data were analyzed by using one-way ANOVA (SAS Institute, Cary, NC) with genotype as a main factor, followed by a Bonferroni/Dunn post hoc test with correction for multiple comparisons. The effect of genotype was highly significant (P < 0.0001). Pair-wise comparisons revealed the significance of the differences, as follows: P < 0.0001 between tg+A-/- and A+/-, tg+A-/- and A+/-Rh+/-, tg+A-/- and A+/+Rh+/-, tg+A-/- and WT, tg+A+/+ and A+/-, tg+A+/+ and A+/-Rh+/-, tg+A+/+ and Rh+/-, tg+A+/+ and WT, A+/- and A+/+Rh+/-, A+/-Rh+/- and A+/+Rh+/-A+/-Rh+/- and WT, A+/+Rh+/- and WT; P = 0.0005 between tg+A-/- and tg+A+/+; P = 0.0007 between A+/- and WT. The difference between A+/- and A+/-Rh+/- was not significant (P = 0.8394).

Quantification of Purified Proteins. The purity of proteins in preparations used for this study (bovine and mouse rod arrestin and rhodopsin) was >95%, as determined by SDS/PAGE, followed by Coomassie blue staining. Initial protein quantification by the Bradford method (Bio-Rad) was confirmed by amino acid analysis performed by the Protein Chemistry Core Lab (Baylor College of Medicine). To this end, each protein (in two aliquots containing 6-10 mg) was precipitated by the addition of nine volumes of methanol (to remove buffer components as well as lipids from the rhodopsin sample) and pelleted by centrifugation for 10 min at 13,000 ´g in 6 ´ 50-mm borosilicate tubes. The pellets were washed with 0.2 ml of 90% methanol and dried. The proteins were hydrolyzed with HCl (vapor phase, 150°C, 75 min) along with the standard amino acid mix (to control for the loss of amino acids under these conditions). Samples were dissolved in 100 ml of water, and 10- and 20-ml aliquots of each sample were analyzed in duplicate. The quantification of amino acids stable under the conditions of acid hydrolysis (Ala, Val, Leu, Phe, and Lys) was used to calculate the amount of each protein in the sample based on its known amino acid composition.

Quantitative Western Blot. Aliquots of eyecup homogenates containing 0.1-1.5 ng of rhodopsin, 10-100 pg of arrestin, or 0.1-1 ng of tubulin were subjected to SDS/PAGE along with four to six standards containing known amounts of the corresponding proteins (quantified by amino acid analysis) in the same range. Rhodopsin and arrestin standards were supplemented with the same amount of protein from the retina of rhodopsin and arrestin knockout mice, respectively, that was present in experimental samples. The proteins were transferred to an Immobilon-P (Millipore) membrane, which was blocked with 5% nonfat dry milk in TBS containing 0.1% Tween 20 (TBST) for 30-60 min at 37°C with gentle rocking. The membranes were rinsed with TBST, and arrestin, rhodopsin, and tubulin blots were incubated overnight at 4°C with gentle rocking with monoclonal F4C1 anti-arrestin antibody (1:10,000) (a generous gift from L.A. Donoso, Wills Eye Hospital, Philadelphia, PA), monoclonal 4D2 anti-rhodopsin antibody (1:3,000) (a generous gift from R. S. Molday, University of British Columbia, BC, Canada), and monoclonal D66 anti-b-tubulin antibody (1:5,000) (ABR-Affinity Bio-Reagents) in TBST supplemented with 2% BSA. Unbound primary antibodies were removed by several washes with TBST, and the blots were incubated with HRP-conjugated anti-mouse secondary antibodies (1:10,000) (Jackson ImmunoResearch) and washed, and the bands were visualized with WestPico chemiluminescence reagent (Pierce) according to manufacturer's instructions. Developed blots were exposed to SuperRX x-ray film (FujiFilm) for appropriate periods to ensure that none of the bands to be used for quantification purposes are saturated on the film. The signals were quantified by using VersaDoc with QuantityOne software (BioRad), and the amount of arrestin, rhodopsin, or tubulin in experimental samples was calculated based on linear calibration curves (SI Fig. 3), by using GraphPad Prizm software. Because rhodopsin always appears as a series of bands believed to represent monomers, dimers, trimers, and higher-order oligomers (SI Fig. 3A), the sum of the signal in all rhodopsin bands in each lane was used for quantification. For rhodopsin and arrestin quantification, samples containing equivalent amounts of retinal protein from rhodopsin and arrestin knockout mice, respectively, was run on each blot, and the signal in corresponding areas of these "blank" lanes was subtracted from the signal in standards and all other samples. Each protein in every eye sample was quantified on two to three independent blots.

This Article

  1. Published online before print February 20, 2007, doi: 10.1073/pnas.0610886104 PNAS February 27, 2007 vol. 104 no. 9 3125-3128
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