FIGURE 8.

P-Rex1 and Norbin promote the localization of each other in membrane fractions. A, HEK-293 cells expressing EE-P-Rex1 and/or myc-Norbin, and mock-transfected cells, were serum-starved, lysed, the total lysates were cleared of debris and nuclei, and the postnuclear supernatant fractionated into membrane and cytosol fractions. Aliquots from all stages of the fractionation were analyzed by Western blotting with P-Rex1 6F12 and Norbin C1 antibodies. Note that 5 times as much membrane fraction was loaded than of the other fractions, to allow direct comparison. Blots shown are from 1 experiment representative of 3. Total lysates and postnuclear supernatant were run on one gel, cytosol and membrane fractions on another; the vertical white lines denote cropped lanes. B and C, Western blots as in A were quantified by ImageJ and the amount of P-Rex1 (B) and Norbin (C) in the membrane fraction expressed as percent of the postnuclear supernatant. Data are mean ± S.E. from 3 independent experiments; statistics are unpaired t test.