Immobilon chemiluminescence substrate

Cells were lysed by agitation on a rotating platform at 4°C for 1 hour in lysis buffer containing 50 mM Tris/ HCl (pH 7.5), 150 mM NaCl, 1% NP40, 0.5% deoxycholic acid, 0.1% sodium dodecyl sulfate (SDS), 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride , and 10 mg/mL aprotinin. Cellular debris was removed by centrifugation at 16,060 × g and 4°C for 20 min. An equal amount of protein of each cell extracts was subjected to 10% SDS-PAGE and the proteins were transferred using semi-dry blotting to Hybond-ECL membranes (GE Healthcare, Glattbrugg, Switzerland). Western blot analysis was performed by probing the membrane with the following antibodies at the indicated dilution as follows: anti-rabbit ΔNp63 (1:500; provided by Dr. James DiRenzo, Dartmouth Medical School), anti- goat GLI2 (1:200, Santa Cruz Biotechnology, CA, USA), anti-rabbit GAPDH (1:3000; Santa Cruz Biotechnology) and species specific horseradish peroxidase conjugated secondary antibodies (Santa Cruz Biotechnology). Peroxidase activity was detected using the Immobilon chemiluminescence substrate (Millipore, Billerica, MA, USA) and the signals were recorded using a VersaDoc Imaging System (Bio-Rad, Hercules, CA, USA).

Cell culture flasks were coated with 125 μg/ml rat tail collagen (Sigma-Aldrich) diluted in 100 mm CH₃COOH for at least 2 h prior to washout with PBS and cell plating. WKPT cells (3 × 10⁶/75 cm²) were grown to confluence and treated with 0.1 mm DFO for 16 h in normal culture medium. Surface biotinylation on the adherent monolayer was performed using a Cell Surface Biotinylation kit (Pierce) according to the manufacturer's instructions. Homogenate samples were collected after the cell lysis step. Protein concentration was determined by the method of Lowry et al. (24 (link)). Equal amounts of protein were separated by SDS-PAGE, transferred to polyvinylidene fluoride membranes, and immunoblotted using mouse anti-TfR1 (clone H68.4; 1:5000; Thermo Fisher), rabbit anti-Na⁺/K⁺-ATPase (1:1000; Cell Signaling Technology), rabbit anti-GAPDH (1:40,000; Cell Signaling Technology), and species-specific horseradish peroxidase–coupled secondary antibodies (Jackson ImmunoResearch Laboratories). Signals were visualized using Immobilon chemiluminescence substrate (Millipore) and blue X-ray films. Densitometry analysis was performed using ImageJ/Fiji. Protein loading was controlled by Coomassie blue gel staining.

Protein concentration of nuclear and cytoplasmic protein fractions was determined using the DC Protein Assay (Bio-Rad). Nuclear and/or cytoplasmic fractions were solubilized in Laemmli buffer (Bio-Rad) with 5% β-mercaptoethanol (Bio-Rad) and separated by size using NuPAGE 4-12% Bis-Tris gels with MES SDS running buffer, alongside PageRuler Plus pre-stained protein ladder (all Life Technologies). Proteins were transferred to PVDF membrane using NuPAGE transfer buffer (Life Technologies) with 10% ethanol (Sigma-Aldrich). Membranes were blocked using 5% w/v non-fat milk powder in Tris-buffered saline with 0.05% Tween-20 (Sigma-Aldrich) (TBS-T), then incubated with primary antibodies overnight at 4°C (Table S1), washed in TBS-T, incubated for 1 h with secondary antibody at room temperature (Table S1) and washed in TBS-T. Membranes were then imaged using Amersham Imager 600 (GE Lifesciences) with Immobilon chemiluminescence substrate (Millipore). Membranes were sequentially re-probed after incubation in Restore PLUS Western Blot Stripping Buffer (Thermo Fisher Scientific). Quantification of western blot bands was performed with integrated density values obtained using ImageJ.