Restore western blot stripping buffer

Dulbecco’s Modified Essential Medium (DMEM), penicillin, streptomyosin, Alexa Fluor 488 phalloidin and RNase A were purchased from Invitrogen (Carlsbad, CA). Roswell Park Memorial Institute (RPMI) 1640 was purchased from Sigma (St. Louis, MO). Restore Western Blot Stripping Buffer and NE-PER Nuclear and Cytoplasmic Extraction Kit were purchased from Thermo Scientific (Waltham, MA), fetal bovine serum from Hyclone (Logan, UT), Restore Western Blot Stripping Buffer and NE-PER Nuclear and Cytoplasmic Extraction Kit from Thermo Scientific (Waltham, MA). Poly (ε -caprolactone) (PCL, [(CH2)5COO]n-), having 80,000 MW, was purchased from Sigma Aldrich, Inc. The solvent used for electrospinning was methylene chloride (density = 1.32g/cm³, boiling point = 39.75°C, dielectric constant = 9.1).

The cells were incubated with CytoBuster^TM Protein Extraction Reagent (Novagen, Darmstadt, Germany), protease inhibitor cocktail (Sigma-Aldrich, MO, USA) and phosphatase inhibitor cocktail 2 (Sigma-Aldrich) for 30 min at 4 °C. The cell extracts were then purified by centrifugation at 12,000 rpm for 15 min at 4 °C, and protein concentrations were determined by Coomassie Plus^TM Protein Assay Reagent (Thermo Fisher Scientific). Protein samples (15 μg) were mixed with Tricine sample buffer (Protech Technology, Taipei, Taiwan), heated for 5 min at 95 °C, resolved on a 4–12% Bis/Tris NuPAGE gel (Invitrogen, Carlsbad, CA, USA) and transferred to a Nitrocellulose blotting membrane (Amersham Biosciences, Piscataway, NJ, USA) using a Trans-Blot SD Semi-Dry Electrophoretic Transfer Cell (Bio-Rad Laboratories, Hercules, CA, USA). The membranes was subsequently blocked with 5% Difco^TM Skim Milk (BD Biosciences, Franklin Lakes, NJ, USA) in PBS for 1 h, hybridized with the indicated Ab and then incubated with horseradish peroxidase (HRP)-conjugated sheep anti-mouse IgG or goat anti-rabbit IgG (Millipore, Billerica, MA, USA) for 1 h before being incubated with Immobilon^TM Western Chemiluminescent HRP Substrate (Millipore). The immuno-reactive signals were stripped by Restore^TM Western Blot Stripping Buffer (Thermo Fisher Scientific) before the protein of interest was re-probed.

Proteins from cell lysates (20–60 μg), and tissue samples (20–70 µg) were separated on a 10% or 10–20% gradient acrylamide gel and then transferred to a 0.45 μM pore PVDF membrane (Merck, Darmstadt, Germany) as previously described [57 (link)]. The primary antibodies used are shown in Supplemental Table S4 and all of them were diluted in TTBS. Rabbit and mouse primary antibodies were immunodetected using horseradish peroxidase-conjugated anti-rabbit IgG (NA931V; 1:4000 in TTBS) or anti-mouse IgG secondary antibody (NA934V; 1:5000 in TTBS) (GE Healthcare, Buckinghamshire, UK), respectively. When possible, phospho-proteins and their total expression were detected in the same gel, using Restore^TM Western Blot Stripping Buffer (Thermo Fisher Scientific) as per the manufacturer’s instructions, blocking the membrane again before the incubation with the next antibody. Loading was normalized by β-actin or α-tubulin. Protein bands were visualized using the Clarity Western Blot Analysis ECL (BioRad^®, Hercules, CA, USA). Band densitometry was analysed using ImageJ Software (v1.52a, Wayne Rasband, National Institute of Health, Stapleton, NY, USA).