Results: 4

1.
Figure 4

Figure 4. From: Stretched Extracellular Matrix Proteins Turn Fouling and Are Functionally Rescued by the Chaperones Albumin and Casein.

Colocalization analysis of soluble Fn and the 70 kDa Fn fragment binding to stretched or relaxed fibrillar Fn. The data were analyzed and displayed analogous to the ones shown in Figure 1. (a−d) Full-length Fn and (e−h) is its 70 kD N-terminal fragment.

William C. Little, et al. Nano Lett. 2009 December 9;9(12):4158-4167.
2.
Figure 3

Figure 3. From: Stretched Extracellular Matrix Proteins Turn Fouling and Are Functionally Rescued by the Chaperones Albumin and Casein.

Reversibility of switching the FRET signature when relaxing Fn fibers and the associated release of nonspecifically bound proteins. (a) Fn fibers were manually deposited in parallel orientations onto prestrained silicone substrates and incubated with Alexa 633-labeled BSA or nothing as a control. The substrates were imaged for FRET as well as the relative BSA-633 intensity of the bound proteins before and after the indicated absolute strains factor values (b). After imaging at 5, 10, 20, and 30 min, the samples were restretched to their original conformation and again imaged. Each data point represents (a) the mean ± standard deviation of the average acceptor intensity divided by donor intensity (IA/ID) per pixel, or (b) the mean ± standard deviation of the normalized average BSA-633 intensity of all pixels that contained Fn to distinguish between protein binding to the background silicone substrate.

William C. Little, et al. Nano Lett. 2009 December 9;9(12):4158-4167.
3.
Figure 1

Figure 1. From: Stretched Extracellular Matrix Proteins Turn Fouling and Are Functionally Rescued by the Chaperones Albumin and Casein.

Colocalization analysis of albumin, casein, and the Fn antibody L8 (FnI9-FnIII1) to fibrillar Fn stretched in a uniaxial strain device. (a) FRET IA/ID values in false color for Fn fibers that have incorporated 5% of Fn-DA. (b) Corresponding histogram with calibration lines indicating the IA/ID ratios of soluble Fn denatured by GdnHCl in solution. (c) Fluorescence intensity of BSA labeled with Alexa Fluor 633 (BSA-633) adsorbed to Fn fibers. Absolute fiber strains shown were defined as 100(L-Lo)/Lo, where L is the extended fiber length and Lo is the fiber length when fully relaxed. (d) The corresponding plot giving the BSA-633 intensity normalized per labeled Fn. The color bar represents the number of pixels with a given IA/ID ratio of fibrillar Fn and BSA intensity. The medians are given as white dots. Panels (e−h) and (i−l) show the same data anlysis displayed for casein and Fn antibody L8, respectively. Data averages obtained from 10 fields of views for albumin, casein and for all proteins in whole bovine serum are shown in the Supplements (Figure S4). (Please note that our FRET ratios were not corrected for minute crosstalk between the excitation and emission channels, since absolute distances were not calculated here.)

William C. Little, et al. Nano Lett. 2009 December 9;9(12):4158-4167.
4.
Figure 2

Figure 2. From: Stretched Extracellular Matrix Proteins Turn Fouling and Are Functionally Rescued by the Chaperones Albumin and Casein.

Force-induced exposure of cryptic cysteines on FnIII7 and FnIII15 evaluated with iodoacetamide and bis-ANS binding to Fn fibers physisorbed to silicone sheets. (a) Fibers were pulled from a drop of wild-type Fn (blue) and deposited onto a silicone sheet that was mounted into a uniaxial stretch device.(14) As a negative control, a drop of alkylated Fn (red) randomly labeled with Alexa 633 on lysines was positioned at the opposite side, and fibers were pulled and deposited in between fibers of alkylated Fn. (b) Differential interference contrast image of prepared fiber sample after it had been washed and stretched in BPS buffer containing 2% BSA. (c) Fluorescence emission of Alexa 633 conjugated to 5% of the fibrillar Fn molecules. (d) Alexa 488 maleimide binding to stretch-exposed cryptic binding sites. After stretching, 5 ng of Alexa 488 maleimide were added to the solution and allowed to react for 15 min. (e−i) Differentially strained Fn fibers that were deposited under various angles to the strain axis. After deposition, all exposed hydrophobic patches on these fibers were blocked with iodoacetamide. Data analysis was performed by calculating the Alexa 488 to Fn-633 ratios. (j−k) Stretch-dependent binding of bis-ANS to Fn fibers. Fn fibers containing 5% Alexa Fluor 633-labeled Fn (Fn-633) were manually deposited onto prestretched silicon sheets in various orientations and subjected to a 0 and a 300% absolute strain, as indicated. Bis-ANS was then adsorbed for 45 min to the substrates, and the ratio of bis-ANS over the Fn-633 intensity was calculated for each pixel ((j) inset histogram). The intensity ratio was converted into a false shade of blues, where lighter colors indicated a greater degree of bis-ANS binding, and was displayed in the spatially resolved representative image shown. (k) The intensity ratio of the bix-ANS over the Fn-633 fluorescence, computed pixel-by-pixel, for 48 fibers oriented in various directions versus their absolute strain. Scale bar in (b) is 100 μm.

William C. Little, et al. Nano Lett. 2009 December 9;9(12):4158-4167.

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