Western Blotting Protein Analysis

Proteins were extracted from snap-frozen tissues and cell pellets, and western blotting (WB) was performed using Bolt 4–12% Bis-Tris gels (cat# NW04125BOX, ThermoFisher Scientific), an iBlot 2 apparatus (cat# IB21001, ThermoFisher Scientific) and an iBind apparatus (cat# SLF1000 or SLF2000, ThermoFisher Scientific), as described [27 (link)]. Primary antibodies used are outlined in Table 2. Secondary antibodies included: HRP-linked goat anti-rabbit (1:1000; cat# ab6721, Abcam), HRP-linked goat anti-rabbit (1:1000; cat# 111-035-045, Jackson Immunology), and Alexa Fluor^® 488 donkey anti-mouse (1:1000; cat# A-21202, ThermoFisher Scientific).
Membranes were imaged using a ChemiDoc MP Imaging System (Biorad) and ImageLab 6.0 software (Biorad). Densitometry was performed using ImageLab 6.0, with the intensity values for the protein-of-interest normalized against α-tubulin. Densitometry data were analyzed using GraphPad Prism 8 (San Diego, CA, USA).