Fig. 4.
λ-Carrageenan modulation of occludin trafficking. Rat cerebral microvessels prepared from brains from either saline-injected or λ-carrageenan-injected animals were homogenized in a neutral pH, detergent-free buffer (containing MgCl2 and CaCl2) and fractionated in a discontinuous 0–20% OptiPrep gradient (in the absence of MgCl2 and CaCl2). One-millilitre fractions were collected from the top of the gradient. (a) Western blots of the first 15 fractions from a representative gradient (for each animal treatment) probed for occludin. Samples were electrophoresed in the presence of 2% sodium dodecyl sulfate and the hydrophilic reducing agent, tris (2-carboxyethyl) phosphine hydrochloride (TCEP), to determine relative distribution of TCEP-resistant (TCEP-R) occludin isoforms. Bars at the top of the figure indicate the distribution of lipid raft and non-raft markers on the gradient. (b) Histograms showing relative distribution, for each animal treatment, of each type of occludin isoform calculated as a percentage of the total band density for that isoform across the gradient. (c) Relative amounts of occludin isoforms detected in fractions 7 and 8, for each animal treatment, calculated as a percentage of the sum of band densities for all isoforms within the same fraction. (d) Graphs showing relationship between fraction density (open circles) and TCEP-R occludin oligomer/dimer ratio (closed circles) calculated for fractions 7, 8, and 10. Data shown are representative of three independent experiments (n = 15 rats) for each treatment condition. LMW, low molecular weight; Sal, saline; Carr, carrageenan.