FIGURE 3.

aPKCι is required for Tyr-tubulin membrane association. A, quantitative membrane binding assay was performed as described under “Experimental Procedures.” Salt-washed HeLa microsomes were incubated without or with 75 or 150 ng of purified recombinant Rab2 N′Δ19, cytosol, and GTPγS for 12 min at 32 °C, as described under “Experimental Procedures.” The membrane pellet was subjected to SDS-PAGE and transferred to nitrocellulose, and the blot was probed with anti-Tyr-tubulin. A representative Western blot from one of three independent experiments is shown. cont, control. B, salt-washed HeLa microsomes were incubated without or with 100 or 200 ng of purified recombinant aPKCι and incubated as above. The membrane pellet was processed as above, and the blot was probed with anti-Tyr-tubulin and with anti-Glu-tubulin. The pool indicates total Tyr- and Glu-tubulin present in the cytosol added to each binding reaction. Results are presented as percent of total pool for each tubulin subtype. A representative Western blot from one of three independent experiments is shown. C, salt-washed HeLa microsomes were incubated without or with 150 ng of purified recombinant Rab2, cytosol, and GTPγS in the presence or absence of increasing concentrations of anti-PKCι for 12 min at 32 °C. The membrane pellet was processed as above, and the blot was probed with the specific and indicated primary antibodies. A representative Western blot from one of three independent experiments is shown.