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. 2019 Dec 20;12(1):30. doi: 10.3390/cancers12010030

Figure 1.

Figure 1

Identification of Galectin-8 as K-Ras interaction partner. (A)Identification of Galectin-8 by mass spectrometry. Purified, post-translationally modified HA-H-Ras (G12V), HA-K-Ras (G12V), and HA-N-Ras (G12V) (500 ng) immobilized on anti-HA µMacs beads were incubated with PANC-1 lysate and precipitates were separated by SDS-PAGE. As controls, PANC-1 lysate were incubated with anti-HA microbeads only [PANC-1 lysate] and HA-K-Ras (G12V) were coupled microbeads only [HA-K-Ras (G12V)]. Peptides from the indicated proteins (*A–*D, and *K1, *K2 as controls, * = extracted protein band), extracted from the silver-stained gel were analyzed by ESI-MS⁄MS mass spectrometry (n = 3). The table shows the identified proteins. (B) Detection of precipitated Galectin-8. HA-Ras interacting proteins were prepared as outlined in (A) and analyzed by western blot. The upper panel shows the detection of Galectin-8 while using anti-Gal-8 antibody and the lower panel the corresponding silver-stained gel of one third of the eluted proteins to control for equal precipitation of Ras isoforms (n = 3). (C) Interaction of EGFP-tagged Ras isoforms with Galectin-8. EGFP-tagged K-Ras (G12V), H-Ras (G12V), or N-Ras (G12V) were transiently expressed in HEK293 cell, immunoprecipitated using anti-GFP µMacs beads and then incubated with PANC-1 lysate for interaction assays. The precipitates were analyzed by western blotting and rec.Gal-8s (30 ng) was applied as a control. Upper panel: detection of co-precipitated Galectin-8; middle panel: precipitated EGFP Ras isoforms; lower panel: endogenous Galectin-8 in 80 µg of the PANC-1 lysates (n = 4). (D) Co-immunoprecipitation of endogenous K-Ras with Galectin-8. Endogenous K-Ras was precipitated from PANC-1 cell lysate (8 mg) while using anti-K-Ras2B antibody [anti-K-Ras] and protein A µMacs beads. Precipitation with rabbit IgG [control IgG] was performed as control. The precipitates were analyzed by western blotting. The amount of Galectin-8 and K-Ras was controlled in 1/150th of the lysate [lysate input], and 10 ng rec.Gal-8s served as a control. Upper panel: Co-precipitated Galectin-8; lower panel: precipitated K-Ras (n = 3). (E) The interaction of purified K-Ras and Galectin-8. The indicated amounts of His-K-Ras (G12V) [H5/His-K-Ras] or H5/His-Gal-8l, each expressed in H5 insect cells (left panel), and indicated amounts of E. coli-derived His-K-Ras [E. coli/His-K-Ras] and E. coli-derived Galectin-8 short [rec.Gal-8s] (right panel) were dotted as baits onto nitrocellulose membranes. Rabbit IgG served as control. The membranes were either incubated with 1 µg of rec.Gal-8s or with 1 µg HA-K-Ras (G12V) from Sf9 insect cells as a prey. Bound (upper blots) or spotted (lower blots) proteins were detected by the indicated antibodies (n = 2).