Horseradish peroxidase conjugated anti mouse or anti rabbit secondary antibody

SkMel147 melanoma cells were lysed in ice-cold RIPA buffer supplemented with protease inhibitor cocktail (Roche), centrifuged at 14,000 rpm at 4 °C for 20 min and the supernatant was collected. Protein in the supernatant was estimated by DC protein assay (Bio-rad). Totally, 20 µg of protein were resolved by NuPage 4–12% Bis–Tris (Invitrogen) and transferred onto Immobilon-P polyvinylidene fluoride membranes (Millipore). Membranes were washed with distilled water followed by blocking with 5% nonfat dry milk in Tris-buffered saline supplemented with 0.05% Tween-20 (TBST) for 2 h at room temperature. Membranes were washed briefly with TBST then incubated in anti-ZNF180 (1:1000; Sigma SAB1306465), and anti-Tubulin (1:5000; Sigma, T9026) primary antibodies diluted in 5% nonfat dry milk in TBST (0.05% Tween-20) and incubated on a plate shaker for overnight at 4 °C. Membranes were washed for 3 times with TBST followed by incubation with horseradish peroxidase-conjugated anti-mouse or anti-rabbit secondary antibodies (1:5000; Sigma). Membranes were washed three times with TBST then signals were detected using Clarity Western ECL Blotting Substrate (BioRad) and imaged in LICOR Odyssey Fc imaging system.

Fractionation of nuclear and cytoplasmic fractions was done with the NE-PER kit (Thermo Scientific). Protein samples were separated by 15% SDS-PAGE and then electroblotted onto nitrocellulose membranes (Amersham Protran 0.2 NC, GE Healthcare), blocked with 5% non-fat dry milk in Tris-buffered saline +0.1% Tween (TBS-T). Probing of the blots was done with monoclonal anti-FLAG (F7425, Sigma) in TBS-T and 5% non-fat dry milk, or anti-DDX5 (D15E10, Cell Signaling) in TBS-T, washed three times in TBS-T and 5% non-fat dry milk, followed by incubation with horseradish-peroxidase-conjugated anti-mouse or anti-rabbit secondary antibodies (Sigma) for 2 hr in TBS-T and 5% non-fat dry milk. Protein signals were detected with chemiluminescence imaging (Amersham Imager 600RGB).

Protein samples were resolved by SDS-PAGE on 8% polyacrylamide gels and transferred to nitrocellulose. Monoclonal mouse anti-FLAG (M2, Sigma) antibodies were used at a dilution of 1:5,000. Polyclonal rabbit Pmt4- (22 (link)) and Sec61-directed (47 (link)) antibodies were used at a dilution of 1:2,500 and 1:1,000, respectively. Blots were incubated with horseradish peroxidase-conjugated anti-mouse or anti-rabbit secondary antibodies (Sigma). Protein-antibody complexes were visualized by enhanced chemiluminescence and quantified with the ImageQuant LAS 4000 imaging system (GE Healthcare).

HUH7 cells (1.5×10⁵ per well) were seeded onto 6-well plates and the day after the culture medium was exchanged to FBS-free medium. After 12 hours the cells were treated with increasing doses of BMP-6 (60; 200; 800 ng/mL; R&D Systems) and/or IL-6 (2; 4; 6; 25 ng/mL; R&D Systems) for 12 hours and then harvested for protein analysis. Cells were washed twice in ice cold Dulbecco's phosphate-buffered saline (PBS). Cell pellets were lysed in ice-cold NET buffer (1% Triton X-100 (v/v), 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 5 mM EDTA, 20 mM NaF, 1 mM Na₃VO₄) supplemented with 1× Complete Mini Protease Inhibitor Mixture (Complete Mini,Roche Applied Science). The protein concentration was measured using the BCA (bicinchoninic acid) Protein Assay (Pierce). Protein lysates (15 µg) were subjected to 10% SDS-PAGE and transferred to a nitrocellulose membrane (Whatman) for protein immunodetection using rabbit anti-phospho SMAD 1/5/8 (Cell Signaling #9511), mouse anti-phospho STAT3 (Cell Signaling, #9138) and mouse anti-actin (Sigma Aldrich, A2228). Blots were then incubated with horseradish peroxidase conjugated anti-mouse or anti-rabbit secondary antibodies (Sigma Aldrich) and then subjected to chemiluminescence (Amersham Biosciences, ECL Plus). For the densitometric analysis the resulting bands were digitalized and quantified using the NIH Image J software (rsb.info.nih.gov/nih-image/)