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

Figure 2.

Figure 2

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).