Ep1553y

Cochleae were dissected from newborn (postnatal day 0, P0), P6, P12, P25 and P90 mice. After rapid decapitation, inner ears were removed and fixed for 2h in 4% formaldehyde. If necessary, tissues were decalcified prior to cryosectioning. Sections were cut parallel to the modiolus (mid-modiolar cut). Tissue sections of the cochlea were incubated overnight at 4 °C with primary antibodies directed against LDLR (rabbit monoclonal antibody [EP1553Y]; 1:50; Abcam; ab52818), myosin VIIa (mouse monoclonal antibody; 1:50; Santa Cruz Biotechnology; sc-74516), Kir4.1 (mouse monoclonal antibody; 1:50; Santa Cruz Biotechnology; sc-293252) and VE-cadherin (goat polyclonal antibody; 1:50; R&D Systems; AF938). The anti-LDLR antibody used in this study was found to give a positive signal in western-blot performed with liver extracts of wildtype mice, but not with liver extracts of Ldlr -/ -mice ( Li et al., 2021 ) (link). Tissues were then incubated for 1h with Rhodamine Red X-, Cy5-and/or FITC-conjugated anti-mouse, anti-goat and antirabbit IgGs secondary antibodies (Jackson Immunoresearch Laboratories). F-actin staining was obtained using Phalloidin-iFluor 647 reagent (1:50; Abcam; ab176759). Antibody specificity was tested by omitting each of the primary antibodies. Pictures were representative of three age-matched cochleae, from three different animals.

U-87 MG, PANC-1 and CAL-33 tumor cells cultured in 2D were harvested, washed with PBS, lysed and their protein content was quantified using Micro BCA™ Protein Assay Kit (Thermo Fisher Scientific). Forty micrograms of protein/cell line were loaded into a 12% polyacrylamide gel. Following migration/ electrophoresis, the proteins were transferred onto an activated PVDF ( polyvinylidene difluoride) membrane (#10600023, Amersham). The membrane was then blocked with 5% milk for 1 h, at RT, and incubated with primary antibodies overnight at 4 °C: anti-LDL receptor antibody (1/1000, Abcam [EP1553Y] (ab52818)), anti-scavenging receptor SR-BI antibody (1/2000, Abcam [EPR20190] (ab217318)), and anti-GAPDH antibody (1/1000, SC-365062, Santa Cruz Biotechnology) serving as loading and transfer control. The membrane was then incubated with the secondary antibodies, anti-mouse/rabbit-horseradish peroxidase HRP (1/3000, #7076S, Cell Signaling Technology) for 1 h, at RT. The proteins (LDLR/SR-B1/GAPDH) were finally revealed using Clarity Western ECL Substrate (Bio-Rad), and western blot imaging was performed using Fusion FX (Vilber). LDLR/SR-BI bands intensities were quantified and normalized to GAPDH bands in each cell line, using ImageJ software.