Ecl chemiluminescent kit

Forty-eight hours post-transfection, cells were harvested and lysed in RIPA buffer supplemented with 1% protease and phosphatase inhibitor cocktail from Thermo Fisher Scientific (Mississauga, ON, Canada). Cell lysates were centrifuged at maximum velocity for 15 min, pellets were discarded, and supernatants containing protein lysates were collected. Protein concentration was quantified by colorimetric Braford protein assay kit (Bio-Rad, Montreal, QC, Canada). Proteins were run on SDS-PAGE gel and transferred to nitrocellulose membranes. Membranes were blocked in 5% milk in tris-buffered saline (TBS-T) and then incubated overnight at 4 °C with primary antibodies (1:1000 dilution). The next day, membranes were washed and incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies. Membranes were developed using an ECL chemiluminescent kit (GE Life Sciences, Mississauga, ON, Canada) and images were obtained using a Bio-Rad Chemidoc imaging system.

Cells and liver tissues were lysed or homogenized in RIPA buffer containing a cocktail of protease inhibitors (Santa Cruz, CA) according to the manufacturer's instruction. Protein extracts were loaded onto 12% acrylamide gels (Bio-Rad) and transferred onto nitrocellulose membranes. Protein bands were visualized with ECL-chemiluminescent kit (GE Healthcare, Piscataway, NJ). The antibodies against anti-PDGFRα, PDGFRβ, ERK, AKT, phosphorylated ERK (Thr202/Tyr204) and phosphorylated AKT (Ser473) were purchased from Cell Signaling Technology (Danvers, MA). GLI2 and GLI3 antibodies were purchased from Proteintech (Rosemont, IL). The antibodies against β-actin was purchased from Abcam (Cambridge, MA).

Cell lysates were run on 5% or 8% polyacrylamide gels and subjected to Western blotting as described previously [6] (link). Antibodies used for Western blot, confocal immunofluorescence microscopy (IF) and immunoprecipitation were; GFP (ab290), GFP-Sepharose (ab69314) (Abcam), Vinculin (Sigma), Emerin (VP-E602) (Vector Labs), lamin A/C (sc-6215) (Santa Cruz), total β-catenin, active β-catenin clone 8E7 (05-665) (Millipore), nesprin-2 CH3 and nesprin-2 N3 (Immune Systems). N2CH3 peptide blocking experiments were performed as described previously using the peptide KRDLDELKDHLQL (Immune Systems) [6] (link). Filamentous actin was observed by IF using Rhodamine phalloidin (Invitrogen). Secondary antibodies for WB were horseradish peroxidase-conjugated anti mouse (NA931) or anti rabbit (NA94V) antibodies from GE Healthcare. ECL chemiluminescent kit (RPN2132, GE Healthcare) was used for detection according to manufacturer's instructions. Invitrogen anti-mouse Alexa fluor 568 (A11031) and anti-rabbit Alexa fluor 488 (A11034) were used as IF secondary antibodies. For IF cells were cultured on cover slips, fixed in 4% paraformaldehyde (Sigma), permeabilised in 0.5% NP-40 (Sigma) and processed as described previously [6] (link). All images were captured at 63 × magnifications using a Leica SP5 laser scanning confocal microscope.