Chemismart 5000 system

For western blotting, protein samples were resolved on pre-cast NuPage 4–12% bis-Tris acrylamide gradient SDS-PAGE gel (Life Technologies). Proteins were then transferred onto nitrocellulose membrane during 2 h at 800 mA in phosphate transfer buffer. Membranes were blocked 1 h in PBS-0.2% Tween 20, 10% skimmed milk and incubated overnight at 4°C with primary antibodies. Membranes were incubated with the appropriate secondary antibodies coupled to HRP (horse radish phosphatase ) and revealed using West Dura from Pierce (Thermo Scientific) and ChemiSmart 5000 system (Vilber Lourmat) and quantified using Chemicapt software.

Twenty-microgram proteins were separated using 4–20% CRITERION® TGX Stain-Free™ Gels (300 V, 20 min) and subsequently transferred to Trans-Blot® Turbo™ Nitrocellulose membranes (both Bio-Rad, Hercules, CA, USA). The membranes were blocked for 1 h at RT with 5% BSA in TBST (0.1% Tween20), before the primary antibodies were applied diluted 1:1000 in the same buffer (TrkA, Cell Signaling [2510]; pTrkA (Y674/675), Cell Signaling [4621S]; LMNA, Abcam [ab108595]; pLMNA (S22), Abcam [ab138450]; STMN1, abcam [ab52630]; pSTMN1 (S16), Abcam [ab47328]; pSTMN1 (S25), Abcam [ab194752]), except anti-Gapdh (EMD Millipore [MAB374]), which was diluted 1:2000. The membranes were incubated over night at 4 °C, washed with TBST and consecutively incubated with the appropriate secondary antibodies (anti-Rabbit IgG HRP, Thermo Scientific [31460]; anti-Mouse IgG HRP, Thermo Scientific [31430]) for 1 h at RT. Bands were visualized with SuperSignalTM Chemiluminescent Substrate (Thermo Fisher Scientific, Waltham, MA, USA) and documented using a CHEMI SMART 5000 System (Vilber Lourmat, Eberhardzell, Germany).

Cells were lysed in RIPA buffer supplemented with phosphatase and protease inhibitors (Sigma). Whole-cell extracts were separated on a pre-cast NuPage 4–12% acrylamide gradient sodium dodecyl sulphate polyacrylamide gel electrophoresis gel (Invitrogen) and transferred to a nitrocellulose membrane (Thermo Fisher Scientific) in Tris-glycine transfer buffer. Membranes were blocked with 10% milk and incubated overnight at 4 °C with the primary Abs. Membranes were incubated with the appropriate secondary Abs coupled with horseradish peroxydase for 1 h at RT. Membranes were revealed using SuperSignal West Dura Extended Duration Substrate (Thermo Fisher Scientific) and images were taken with ChemiSmart 5000 system (Vilber Lourmat, Collégien, France).