Fig. 1: Localization of hERG1 and of the hERG1/β1 integrin complex in lipid rafts in PDAC cells after cell adhesion on Fibronectin.

A Representative membrane of lipid rafts localization of hERG1/β1 integrin complex in PANC-1 cells. As a control, we also analyzed the distribution of two marker proteins, one of which (caveolin-1) is highly enriched in the raft fractions (4–6) and one (transferrin receptor/CD71) is mainly distributed in the Triton-soluble fractions (9–11). The panels on the right show the densitometric analysis of the fractions of the sucrose gradient. Bars indicate the percentage distribution across the gel of raft fractions 4–6 (Triton X-100 insoluble fractions) and non- rafts fractions 9–11 (Triton X-100 soluble fractions), as detected by scanning densitometric analysis; quantitation of each enriched protein was normalized over the corresponding marker, i.e caveolin-1 for raft fractions and transferrin receptor/CD71 for non-raft fractions. The density of each band in the same gel was analyzed, values were totaled, and then the percent distribution across the gel was detected and reported in the bar graphs on the right as densitometric units (%) for Triton X-100-insoluble fractions (4–6) and Triton X-100-soluble fractions (9–11). B Representative membrane of Triton X-100-insoluble fractions (4–6) and Triton X-100-soluble fractions (9–11) of PANC-1 cells immunoprecipitated with anti-hERG1 mAb and incubated with Cholera Toxin B Subunit Peroxidase to detect GM1 (left panel) and corresponding densiometric quantification of GM1 normalized over hERG1 immunoprecipitates(right panel). Data are representative of three independent experiments (n = 3). a.u.= arbitrary units. C Representative membrane of Triton X-100-insoluble fractions (4–6) and Triton X-100-soluble fractions (9–11) of PANC-1 cells, immunoprecipitated with mAb hERG1 and incubated with anti-Integrin β1 pAb (RM12 Ab) (left panel) and corresponding densiometric quantification of β1 Integrin over hERG1 immunoprecipitates (right panel). Data are representative of three independent experiments (n = 3). a.u.= arbitrary units. D Representative membrane of Triton X-100-insoluble fractions (4–6) and Triton X-100-soluble fractions (9–11) of HEK293-hERG1 cells, immunoprecipitated with mAb hERG1 and incubated with anti-Integrin β1 pAb (RM12 Ab) (left panel) and densiometric quantification β1 Integrin over hERG1 immunoprecipitates (right panel). Data are representative of three independent experiments (n = 3). a.u. = arbitrary units. E Representative membrane of co-IPs between β1 integrin, hERG1, caveolin-1 and flotillin-1 in HEK-hERG1 cells after cell seeding on FN for 90 min (left panel). Total cell proteins were immunoprecipitated with anti-β1 integrin mAb (TS2/16). An IgG isotypic control was employed too. Cells were seeded on BSA or FN coated dishes for 90 min. HEK 293 were used as control. Right panel: densitometric analysis. Protein lysates used for IP quantification, indicated as “inputs”, are reported in figure; protein lysates not used for IP quantification, indicated as “inputs”, are reported in Supplementary Fig. 1 (panel A). Data are representative of three independent experiments (n = 3). a.u. = arbitrary units. F Co-IP between hERG1 and β1 integrin, Co-IP between hERG1, β1 integrin, caveolin-1 and Co-IP between hERG1, β1 integrin, flotillin in normal and PDAC cells, following 90 min adhesion onto BSA or FN. Protein lysates used for IP quantification, indicated as “inputs”, are reported in figure; protein lysates not used for IP quantification, indicated as “inputs”, are reported in Supplementary Fig. 1 (panel B). Total cell proteins were immunoprecipitated with anti-β1 integrin mAb (TS2/16). An IgG isotypic control was employed. Top panel: representative WB of the co-IP; Bottom panel: densitometric analysis. Total lysates indicated as “inputs” are reported in figure. Data are representative of three independent experiments (n = 3). a.u. = arbitrary units. All data are presented as mean values ± s.e.m. Co-IP between hERG1 and β1 integrin: BSA vs FN: **P < 0.01: PANC-1, MiaPaca2, BxPC3; ***P < 0.001: HPDE FN vs PANC-1 FN, MiaPaca2 FN; **P < 0.01: HPDE FN vs BxPC3 FN; ***P < 0.001: RLT-PSC FN vs PANC-1 FN, MiaPaca2 FN; **P < 0.01: RLT-PSC FN FN vs BxPC3 FN. Co-IP between hERG1, β1 integrin, caveolin-1: BSA vs FN: **P < 0.01: PANC-1, MiaPaca2, BxPC3; ***P < 0.001: HPDE FN vs PANC-1 FN, MiaPaca2 FN; **P < 0.01: HPDE FN vs BxPC3 FN; ***P < 0.001: RLT-PSC FN vs PANC-1 FN, MiaPaca2 FN; **P < 0.01: RLT-PSC FN FN vs BxPC3 FN. Co-IP between hERG1, β1 integrin, flotillin: **P < 0.01: PANC-1, MiaPaca2, BxPC3; ***P < 0.001: HPDE FN vs PANC-1 FN, MiaPaca2 FN; **P < 0.01: HPDE FN vs BxPC3 FN; ***P < 0.001: RLT-PSC FN vs PANC-1 FN, MiaPaca2 FN; **P < 0.01: RLT-PSC FN FN vs BxPC3 FN. **P < 0.01, and ***P < 0.001. All data are presented as mean values ± s.e.m. *P < 0.05 and ***P < 0.001 (one-way ANOVA). Insol insoluble fraction, Sol soluble fraction, Input total lysate, BSA bovine serum albumin, FN Fibronectin, St standard.