*4.10. Protein Extraction and Western Blot*

Western blot analysis was performed using whole cell lysate to quantify proteins in cells and tissue samples. For protein extraction, cell pellets were resuspended in 200–400 µL radioimmunoprecipitation assay (RIPA) buffer (20 mM Tris, 137 mM NaCl, 10% glycerol, 1% nonidet *p*-40, 3.5 mM sodium dodecyl sulfate (SDS), 1.2 mM sodium deoxycholate, 1.6 mM ethylenediaminetetraacetic acid (EDTA), pH 8) containing 1x protease/phosphate inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA). Whole cell lysate was incubated on ice for 5 min and then centrifuged at 14,000 g for 5 min to remove cell debris. To extract protein from day 16 TE, the tissue was homogenized in RIPA buffer. Homogenized samples were sonicated using a Bioruptor Sonication System (Diagenode, Denville, NJ, USA) for 5 cycles of 30 s "ON" and 30 s "OFF". Sonicated samples were centrifuged at 14,000 g for 5 min to remove debris. Protein concentration was measured using the bicinchoninic acid (BCA) protein assay kit (ThermoFisher, Waltham, MA, USA). Protein was separated in 4–15% Bis-Tris gels (Bio-Rad Laboratories, Hercules, CA, USA) at 90 volts for 15 min and 125 volts for 60 min, and then transferred to 0.45 µm pore size nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA, USA) at 100 volts for 2 h at 4 ◦C. The membranes were then blocked in 5% non-fat dry milk solution in tris buffered saline with tween 20 (TBST) (50 mM Tris, 150 mM NaCl, 0.05% Tween 20, pH 7.6) for 1 h at room temperature. After blocking, the membranes were washed 3 times with 1x TBST for 5 min each, and then incubated at 4 ◦C overnight with specific primary antibody. After overnight incubation, the membranes were washed 3 times with 1x TBST for 5 min each. After washing, the membranes were incubated with appropriate secondary antibody conjugated to horseradish peroxidase for 1 h at room temperature. After removing the secondary antibody, the membranes were washed following the same procedure and developed using Super Signal WestDura Extended Duration Substrate (ThermoFisher, Waltham, MA, USA) and imaged using ChemiDoc XRS+ chemiluminescence system (Bio-Rad Laboratories, Hercules, CA, USA). The images were quantified using Image-Lab software (Bio-Rad Laboratories, Hercules, CA, USA). To normalize protein quantity, β-actin, α-tubulin, or glyceraldehyde 3-phosphate

dehydrogenase (GAPDH), was used as loading control. Each experiment was repeated on three replicates. The antibodies used and their dilutions are listed in Table S3.
