Mouse α pkcζ h 1

Amylose or glutathione resin was loaded with bacterial lysate (or his purification elutions in the case of Par-3 PDZ1-APM or Par-3 PDZ1-PDZ3 as these proteins contain COOH-terminal his tags) containing MBP-or GST-fusion protein for 30 minutes at 4° C and then washed with wash buffer three times (20 mM HEPES, pH 7.5, 100 mM NaCl, 5 mM MgCl₂, 0.5% Tween 20, and 1 mM DTT). Par-complex was then added to a concentration of 0.5 μM and incubated for 10 minutes at room temperature. In the case where ATP is present, ATP was used at a final concentration of 200 μM in all buffers throughout the pull down experiment and and binding reactions were carried out for 30 minutes at room temperature. Finally, beads were washed two times briefly to remove unbound Par-complex and beads were resuspended in loading dye. Samples were analyzed by SDS-Page and stained by Coomassie as well as Western Blot using α-aPKC (Mouse α-PKCζ H-1 Santa Cruz Biotech sc-17781) and rat α-Par-6.

Amylose resin was loaded with bacterial lysate containing MBP – Par-3 PDZ1 – APM for 30 minutes at 4° C and then washed with wash buffer (20 mM HEPES, pH 7.5, 100 mM NaCl, 5 mM MgCl₂, 200 μM ATP, 0.5% Tween 20, and 1 mM DTT). 2-fold serial dilutions of beads were prepared from 20 μL to 0.625 μL in a total volume of 200 μL. Par-complex was added to a final concentration of 40 nM diluted in wash buffer. After incubation for 30 minutes, beads were collected by centrifugation and an aliquot of supernatant was diluted in loading dye for western blot analysis using α-aPKC (Mouse α-PKCζ H-1 Santa Cruz Biotech sc-17781) and rat α-Par-6. The concentration of protein loaded on the beads was verified by SDS-Page using a standard curve generated with known concentrations of BSA.