Table 2.

Methods for western blot.

Procedure	Protocol
Extraction of proteins from punched tissues	RIPA lysis buffer (25 mM Tris (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, and 0.1% SDS) mixed with protease inhibitor cocktail (one tablet in 10 ml, Roche Diagnostics GmbH, Mannheim, Germany).
Evaluation of the total protein concentration	Bicinchoninic acid (BCA) protein assay (#23228, Thermo Scientific, Rockford, IL) following the manufacturer’s instructions
Separation of an equal amount (30 µg per lane) of protein loaded on each well	SDS-PAGE followed by the electrophoretic transfer of protein bands onto a polyvinylidene difluoride (PVDF) membrane

Detailed description of the Western Blot procedure and protocol used in the present study.