Fig. 3.

Microtubule densification in LVNC myocardial tissues and in 293T cells with HBLV-h-SORBS2 lentivirus transfection. (a) Highly expressed β-tubulin in LVNC hearts compared to normal control hearts by immunofluorescence analyses. The white box in the upper panels indicates the areas enlarged in the lower panels; (scale bars: 25 μm, 10 μm). (b) and (c) The decreased level of free β-tubulin and the increased level of polymerized β-tubulin in LVNC hearts compared to normal control hearts by western blotting analyses. (n = 4 for normal hearts and n = 5 for LVNC hearts, *p < 0.05, **p < 0.01). Data shown as mean ± SEM; a Student's t-test was used for the inferential statistical analysis. (d) Co-localization of SORBS2 (red) and β-tubulin (green) in human cardiac tissues. The co-localized SORBS2 and β-tubulin proteins are visualized in yellow in these human cardiac tissues. The white box in the upper panels represent the areas enlarged in the lower panels (scale bars: 25 μm, 10 μm). (e) An interaction relationship between SORBS2 and β-tubulin in normal control hearts by Co-immunoprecipitation assays. Extracted proteins from all the above mentioned tissues were untreated (input) and the rabbit (IgG) were set as positive and negative controls respectively. SORBS2 antibodies were used for Co-immunoprecipitation (Co-IP), β-tubulin antibody was used or western blot. Likewise, β-tubulin antibodies were used for Co-IP, SORBS2 antibody was used for western blot. Co-immunoprecipitation (Co-IP) of the tissues showing SORBS2 binding with β-tubulin. (f) and (g) Representative images of β-tubulin immunofluorescence staining of 239T cells transfected with HBLV-h-SORBS2 and empty-virus control cells. The expression of β-tubulin in control was decreased compared to the SORBS2 overexpression 293T cells. (n = 25–30 cells per group, **p < 0.01; Student's t-test) (h) and (i) Immunoblotting to assess the β-tubulin level of 239T cells. Quantitative analysis of the expression of β-tubulin in both its free (F) and polymerized (P) forms (n = 3 per group, **p < 0.01; Student's t-test). SORBS2-OE: 293T cells transfected with HBLV-h-3*flag-GFP-SORBS2 lentivirus. Control: 293T cells transfected with HBLV-GFP-PURO-NC lentivirus. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)