Regulation of PYM function by its domains.

<p>(A) Lysates of S2 cells co-expressing HA-eIF4AIII and GFP (control) or GFP-tagged PYM proteins (as indicated at the top of the panel) were immunoprecipitated using protein G (Mock), GFP-Trap (α-GFP IP) and anti-HA (α -HA IP) beads. Input panel shows 1.6% of the extracts and the bound fractions are shown in separate panels. The antibodies used for western analysis are indicated on the right of the panel. The anti-PYM antibody was not used for detection of PYM-GFP proteins due its preferential detection of the <i>Dm</i>PYM C-terminus (data not shown). Arrow and arrowhead indicate endogenous and HA-tagged eIF4AIII proteins, respectively. (B) Lysates of S2 cells expressing GFP (control) or GFP-tagged FL-, ΔC-, or N-PYM proteins were subjected to immunoprecipitation using protein G (Mock) or GFP-Trap (GFP IP) beads under native and DSP cross-linked conditions. 1.6% and 0.32% of inputs utilised in IPs were loaded in lanes 1, 7, 13, 19 and lanes 2, 8, 14, 20, respectively. Bound fractions (20%) were loaded in lanes 4, 10, 16, 22, and the corresponding 5× and 20× dilutions in lanes 5, 11, 17, 23 and lanes 6, 12, 18, 24 respectively. Lanes 3, 9, 15 and 21 contain 20% of the mock IP precipitates. Antibodies utilized for the western blot analysis are indicated at the right of the panels. (C) CoIP efficiencies of Mago and Y14 with GFP (control) or GFP-tagged FL-, ΔC- and N-PYM proteins under native (left panel) and DSP cross-linked (right panel) conditions. Mago and Y14 coIP efficiency is defined as a percentage of measured GFP enrichment in the corresponding GFP IPs. Plotted bar values represent the mean of two biological and four technical replicates.