Supplementary Figure 3: Determination of LINK-A-PIP3 and LINK-A-Ins (1,3,4,5)P4 interactions by RIP and Alpha assay respectively.
(a) Saturation curve used to determine Kd of the interactions between Biotin-Ins (1,3,4,5)P4 and Digoxigenin-labeled full-length LINK-A (left panel), ΔPC LINK-A (middle panel), or ΔPIP3 LINK-A (right panel) in Alpha format (mean ± s.e.m. were derived from n = 3 independent experiments). (b) Competition binding assay to determine Kd for the interactions between biotin- Ins(1,3,4,5)P4 and Digoxigenin-labeled LINK-A in the presence of unlabeled Ins(1,3,4,5)P4 as competitor (mean ± s.e.m. were derived from n = 3 independent experiments). (c and d) Immunoblotting detection (c) or RIP-qPCR detection of indicated RNAs retrieved by PIP3-specific antibody (d) in MDA-MB-231 cells treated with DMSO or LY294002. For a, b and d, mean ± s.e.m. were derived from n = 3 independent experiments (*P < 0.05 and ***P < 0.001, two-tailed paired Student’s t-test). Unprocessed original scans of all blots with size marker are shown in Supplementary Fig. 9.
