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

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© 2019 by the authors.

Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

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Identification of the interacting domains in K-Ras and Galectin-8. (A) Identification of the Galectin-8 carbohydrate recognition domain (CRD) interacting with K-Ras. HEK293 cells were transiently transfected with plasmids encoding for EGFP-K-Ras (G12V) or HA-tagged N-CRD [N], N-CRD-hinge [NH], C-CRD [C], or C-CRD-hinge [CH]. Equal amounts of EGFP-K-Ras were immunoprecipitated and incubated CRD containing lysate. Precipitated proteins were analyzed by western blot. The upper panel shows co-precipitated HA-CRD proteins and the middle panel the immunoprecipitated EGFP-K-Ras proteins. In the lower panel, one-tenth of the HA-tagged CRD-containing lysates were analyzed as an input control. The amount of precipitated HA-CRD proteins was quantified, related to precipitated EGFP-K-Ras and normalized to the amount of HA-N-CRD. The graph shows the interaction index as mean ± SD (*** p ≤ 0.001, n = 3). (B) Interaction of Ras isoforms the CRDs. EGFP-tagged K-Ras(G12V) [K-Ras], H-Ras(G12V) [H-Ras], N-Ras(G12V) [N-Ras], EGFP, as well as HA-N-CRD [N] and HA-N-CRD-hinge [NH] were expressed in HEK293 cells and interaction studies were performed as described in (A). The upper panel represents the detection of co-precipitated HA-N-CRDs, the middle panel the detection of precipitated EGFP-Ras isoforms, and the lower panel the amount of HA-CRD in one-tenth of the lysates (all n = 3). (C) Interaction of Galectin-8 with active and inactive K-Ras. EGFP-tagged, constitutively active K-Ras (G12V) [K-G12V], dominant negative K-Ras (S17N) [K-S17N], and wild-type K-Ras [K-wt] expressed in HEK293 cells were immunoprecipitated and incubated with PANC-1 lysate as a source of endogenous Galectin-8. The precipitates were analyzed by western blot. The upper panel shows the co-precipitated Galectin-8 as well as 30 ng rec.Gal-8s as control. The middle panel presents the precipitated EGFP-K-Ras proteins. In the lower panel the input of Galectin-8 was analyzed in one-twentieth of the applied PANC-1 lysates (n = 5). (D–F) Interaction of Galectin-8 with farnesylated K-Ras. Fully modified EGFP-K-Ras(G12V) [K-G12V], non-farnesylated EGFP-K-Ras(G12V,C185S) [K-G12V,C185S] and EGFP, expressed in HEK293 cells, and unmodified EGFP-K-Ras(G12V) [rec.K-G12V] expressed in E. coli, was precipitated and incubated with (D) 2 mg of PANC-1 cell lysate, (E) 0.5 mg HA-N-CRD, or (F) 0.5 mg HA-C-CRD containing HEK293 cell lysate. The precipitates were analyzed by western blot. The upper panel of each figure shows co-precipitated (D) Galectin-8, (E) HA-N-CRD, and (F) HA-C-CRD. The middle panels illustrate the amount of precipitated EGFP-tagged K-Ras proteins and the lower panels the amount of Galectin-8 and HA-CRD, respectively, in one-tenth of the lysates used (n ≥ 2).