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Figure S1. CERT does not phosphorylate PKD. HEK293T cells were transfected with expression plasmids encoding PKD1-KD-GFP, PKCη-GFP, and Flag-CERT. Cells were lysed in NP-40 extraction buffer containing 1% NP-40 or TEB (substitution of NP-40 with 1% Triton X-100), and Flag-CERT, PKCη-GFP, and PKD-KD-GFP were immunoprecipitated as indicated with Flag- or GFP-specific antibodies. The kinase reaction was performed for 15 min at 37°C in kinase buffer (50 mM Tris, pH 7.4, 10 mM MgCl₂, and 2 mM DTT) in the presence of 2 µCi γ-[³²P]ATP. To terminate the reaction, SDS sample buffer was added, and the samples were resolved by SDS-PAGE, blotted onto nitrocellulose, and analyzed on a phosphorimager (top) followed by immunoblotting with Flag- and GFP-specific antibodies to verify loading (bottom). CERT was only associated with a kinase activity when immunoprecipitates were washed with 1% NP-40-containing buffer. The band marked with an asterisk is the result of nonspecific binding.