egfp h ras  (Addgene inc)

Journal: Cancers

Article Title: Galectin-8 binds to the Farnesylated C-terminus of K-Ras4B and Modifies Ras/ERK Signaling and Migration in Pancreatic and Lung Carcinoma Cells

doi: 10.3390/cancers12010030

Figure Lengend Snippet: 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 Sf 9 insect cells as a prey. Bound (upper blots) or spotted (lower blots) proteins were detected by the indicated antibodies ( n = 2).

Article Snippet: EGFP-H-Ras (G12V) showed an interaction of 33.78 ± 15.14% and EGFP-N-Ras (G12V) of 3.44 ± 5.12% (densitometric analyses of n = 4) when setting the interaction of Galectin-8 short with EGFP-K-Ras (G12V) to 100% as reference.

Techniques: Mass Spectrometry, Purification, Modification, Incubation, SDS Page, Staining, Western Blot, Control, Immunoprecipitation, Derivative Assay

Journal: Cancers

Article Title: Galectin-8 binds to the Farnesylated C-terminus of K-Ras4B and Modifies Ras/ERK Signaling and Migration in Pancreatic and Lung Carcinoma Cells

doi: 10.3390/cancers12010030

Figure Lengend Snippet: Activation of ERK1/2 by downregulation of Galectin-8. ( A – C ). PANC-1 cells and cell clones stably expressing EGFP-K-Ras(G12V) [K-Ras-4.1] or EGFP [EGFP-14] were transiently transfected with Gal-8 [siGal-8] or control [mock] siRNA and lysed 72 h after transfection. The expression of Galectin-8, pERK1/2, ERK1/2, EGFP proteins, and GAPDH was investigated by western blot. 15 ng of rec.Gal-8s was applied as a control. The western blots in ( A ) show the amount of Galectin-8 (upper panel) and of GAPDH as a loading control (lower panel). The bar graph shows the mean ± SEM of densitometric analyses of Galectin-8 long and short in relation to GAPDH, normalized to the mock-transfected control. The values are given as relative intensity (*** p ≤ 0.001, n = 6). ( B ) The phosphorylation of ERK1/2 at Thr202/Tyr204 (upper panel) and the amount of total ERK1/2 (lower panel) was detected simultaneously in Western blot analysis using the Odyssey Infrared Imaging System. The bar graph shows the densitometric analysis of the ratio of pERK1/2 to ERK1/2 normalized to the control (mean ± SEM; * p ≤ 0.05; ** p ≤ 0.01, n = 6). ( C ) Expression of EGFP-K-Ras and EGFP was evaluated using anti-GFP antibody. GAPDH (lower panel) was determined as a loading control. The diagram shows the densitometric analysis as a ratio of EGFP/GAPDH normalized to the mock-transfected control (mean ± SEM; * p ≤ 0.05, n = 6). ( D ) Depletion of Galectin-8 in lung carcinoma cells. Colo699 cells were treated as in (A). The western blots show the amount of Galectin-8 and GAPDH (upper panel) and pERK1/2 and ERK1/2 (lower panel). The diagram shows the densitometric analysis of the ratio of pERK1/2 to ERK1/2 (mean ± SEM; * p ≤ 0.05, n = 4). ( E ) Subcellular localization of EGFP-K-Ras (G12V) and Galectin-8. To determine whether enhanced ERK phosphorylation is associated with an enhanced amount of active, membrane-associated EGFP-K-Ras (G12V), EGFP-14 and K-Ras-4.1 PANC-1 cells were treated as in ( A ). Lysates were fractionated into soluble [S100] (cytosolic) and particulate [P100] (membranous) fractions and they were analyzed by western blot (S100: 50 µg, P100: 25 µg). The upper blot shows the distribution and depletion of Galectin-8 in both fractions, the middle panel displays the distribution of EGFP-K-Ras, especially in the P100 fraction, and the lower panel shows the enrichment of EGFP in the S100 fraction ( n = 4).

Article Snippet: EGFP-H-Ras (G12V) showed an interaction of 33.78 ± 15.14% and EGFP-N-Ras (G12V) of 3.44 ± 5.12% (densitometric analyses of n = 4) when setting the interaction of Galectin-8 short with EGFP-K-Ras (G12V) to 100% as reference.

Techniques: Activation Assay, Clone Assay, Stable Transfection, Expressing, Transfection, Control, Western Blot, Phospho-proteomics, Imaging, Membrane

Journal: Cancers

Article Title: Galectin-8 binds to the Farnesylated C-terminus of K-Ras4B and Modifies Ras/ERK Signaling and Migration in Pancreatic and Lung Carcinoma Cells

doi: 10.3390/cancers12010030

Figure Lengend Snippet: Depletion of Galectin-8 enhances the amount of EGFP-K-Ras and affects ERK. ( A ) PANC-1/EGFP-K-Ras (G12V)-4.1 cells were transiently transfected with increasing amounts of Gal-8 siRNA [siGal-8] or control siRNA [mock]. Protein expression of ERK1/2, pERK1/2, Galectin-8 and EGFP-K-Ras was determined by western blot. The western blots (left) and the diagram (right) show the increasing amounts of EGFP-K-Ras and pERK1/2 as a function of the concentration of siGal-8. The bar graph shows the mean ± SEM of the densitometric analysis (left: EGFP-K-Ras, right: pERK1/2) as relative intensity normalized to the mock-transfected control ( n = 3). ( B ) Increase of EGFP-K-Ras by MG132. PANC-1/EGFP-K-Ras (G12V)-4.1 cells transfected with siGal-8 or control siRNA were left untreated or additionally treated for 24 h or for 48 h with 5 µM MG132. Lysates were prepared 72 h after transfection and then analyzed by western blot. 30 ng of rec.Gal-8s was applied as a control. Detection of β-catenin served as a control for the activity of MG132. GAPDH served as a loading control. The bar graph shows the mean ± SEM of the densitometric analysis of EGFP-K-Ras normalized to the mock-transfected control without MG132 ( n = 3).

Article Snippet: EGFP-H-Ras (G12V) showed an interaction of 33.78 ± 15.14% and EGFP-N-Ras (G12V) of 3.44 ± 5.12% (densitometric analyses of n = 4) when setting the interaction of Galectin-8 short with EGFP-K-Ras (G12V) to 100% as reference.

Techniques: Transfection, Control, Expressing, Western Blot, Concentration Assay, Activity Assay

Journal: Cancers

Article Title: Galectin-8 binds to the Farnesylated C-terminus of K-Ras4B and Modifies Ras/ERK Signaling and Migration in Pancreatic and Lung Carcinoma Cells

doi: 10.3390/cancers12010030

Figure Lengend Snippet: Effects of Galectin-8 depletion on migration and proliferation. ( A ) Wounding assay. PANC-1, EGFP- and EGFP-K-Ras (G12V) expressing cells [PANC-1, EGFP-14, K-Ras-4.1, K-Ras-4.4], and A549 lung cells were transfected with siGal-8 or control siRNA [mock]. After treatment with mitomycin-C, the confluent cell layer was scratched, and images of six randomly chosen points were recorded every 3 h for 48 h. The velocity [µm/h] was calculated, normalized to the mock transfected control of each cell line, and presented as the mean ± SEM (* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001, n = 3–4). The insets below show Galectin-8 depletion 48 h after wounding. The image on the right shows representative phase contrast images of PANC-1 cells. The line represents the wound at t 0h , bar: 100 µm. ( B ) Proliferation. PANC-1 cells and cell clones [PANC-1, EGFP-14, K-Ras-4.1] were transfected with siGal-8 or control siRNA [mock], reseeded after 24 h and images of 5–6 randomly chosen areas were recorded for three days. The bar graph shows the evaluation of the doubling time as mean ± SEM (* p ≤ 0.05, n = 4). The western blot on the right shows the Galectin-8 amount 48 h after transfection.

Article Snippet: EGFP-H-Ras (G12V) showed an interaction of 33.78 ± 15.14% and EGFP-N-Ras (G12V) of 3.44 ± 5.12% (densitometric analyses of n = 4) when setting the interaction of Galectin-8 short with EGFP-K-Ras (G12V) to 100% as reference.

Techniques: Migration, Expressing, Transfection, Control, Clone Assay, Western Blot

Journal: Cancers

Article Title: Galectin-8 binds to the Farnesylated C-terminus of K-Ras4B and Modifies Ras/ERK Signaling and Migration in Pancreatic and Lung Carcinoma Cells

doi: 10.3390/cancers12010030

Figure Lengend Snippet: 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).

Article Snippet: EGFP-H-Ras (G12V) showed an interaction of 33.78 ± 15.14% and EGFP-N-Ras (G12V) of 3.44 ± 5.12% (densitometric analyses of n = 4) when setting the interaction of Galectin-8 short with EGFP-K-Ras (G12V) to 100% as reference.

Techniques: Transfection, Immunoprecipitation, Incubation, Western Blot, Control, Dominant Negative Mutation, Modification

Journal: Cancers

Article Title: Galectin-8 binds to the Farnesylated C-terminus of K-Ras4B and Modifies Ras/ERK Signaling and Migration in Pancreatic and Lung Carcinoma Cells

doi: 10.3390/cancers12010030

Figure Lengend Snippet: Influence of lysine residues of Ras for the interaction with Galectin-8. Ras mutants were expressed in HEK293 cells: ( A ) K-Ras mutants: [K-G12V], [K-G12V,K184P], [K-G12V,K182S], [K-G12V,K182S,K184P] ( B ) H-Ras mutants: [H-G12V], [H-G12V,K185P], [H-G12V,S183K] ( C ) N-Ras mutants: [N-G12V], [N-G12V,P185K], or [N-G12V,G183K,P185K] (see for a detailed description). Co-precipitation assays were performed with PANC-1 cell lysates and precipitates were analyzed by western blot. 30 ng of rec.Gal-8s was used as a control. The upper panels ( A – C ) show co-precipitated Galectin-8 and the middle panels illustrate immunoprecipitated EGFP-Ras proteins. The lower blots show Galectin-8 in 1/25th of the PANC-1 lysates as an input control. The bar graphs document the amount of precipitated Galectin-8 long (black) and short (white), related to the amount of the precipitated EGFP-Ras mutants, and normalized to K-Ras(G12V) (in A ) or H-Ras(G12V) (in B ), respectively. The interaction index was calculated with regard to the interaction of Galectin-8 short with N-Ras (G12V,G183,P185K) due to the very low interaction of Galectin-8 with N-Ras(G12V) (in C ). Each graph shows the mean ± SD of the interaction index (* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001, n = 4–5).

Article Snippet: EGFP-H-Ras (G12V) showed an interaction of 33.78 ± 15.14% and EGFP-N-Ras (G12V) of 3.44 ± 5.12% (densitometric analyses of n = 4) when setting the interaction of Galectin-8 short with EGFP-K-Ras (G12V) to 100% as reference.

Techniques: Western Blot, Control, Immunoprecipitation

Journal: Cancers

Article Title: Galectin-8 binds to the Farnesylated C-terminus of K-Ras4B and Modifies Ras/ERK Signaling and Migration in Pancreatic and Lung Carcinoma Cells

doi: 10.3390/cancers12010030

Figure Lengend Snippet: Influence of the polybasic domain of K-Ras for the interaction with Galectin-8. ( A ) The following K-Ras mutants were expressed in HEK293 cells: EGFP-tagged K-Ras(G12V) [G12V], K-Ras(G12V,K182S,K184P) [K182S,K184P] and mutants in which the six lysines KKKKKK175-180 were replaced by the corresponding amino acids ESGPGC of H-Ras [PL-H], or DGTQGC of N-Ras [PL-N] (see for a detailed description). PANC-1 cell lysates were used for the co-precipitation analyses. Western blot analyzed the precipitates. The upper panel shows co-precipitated Galectin-8, the middle panel displays the immunoprecipitated EGFP-K-Ras mutants and the lower panel shows Galectin-8 in one-twentieth of the PANC-1 lysates used. 30 ng of rec.Gal-8s served as a control. ( B ) The bar graph illustrates the quantification of Galectin-8 long (black) and short (white) in relation to the precipitated EGFP-K-Ras mutants expressed as an interaction index. Each graph shows the mean ± SD (*** p ≤ 0.001, n = 3).

Article Snippet: EGFP-H-Ras (G12V) showed an interaction of 33.78 ± 15.14% and EGFP-N-Ras (G12V) of 3.44 ± 5.12% (densitometric analyses of n = 4) when setting the interaction of Galectin-8 short with EGFP-K-Ras (G12V) to 100% as reference.

Techniques: Western Blot, Immunoprecipitation, Control