Immunoblot Analysis of Cell Lines

CAFs (2 × 10⁴ cells per 12-well dish), Hep3b and SNU423 (5 × 10⁵ cells per 60 mm dish) and SNU398, SNU449, HLF, Huh7 and PLC/PRF5 (4 × 10⁵ cells per 60 mm dish), after the specified treatments, were washed in ice-cold phosphate buffer saline (PBS), pH 7.4, lysed and analyzed by immunoblot as described^{20 (link)}, with antibodies at the following dilutions: fibronectin, 1:10,000 (Sigma-Aldrich, Stockholm, Sweden, F3648); fatty acid synthase (FASN), 1:1,000 (ab22759); calponin, 1:2,000 (EP798Y, ab46794); LXRα, 1:1,000 (ab41902); Smad3, 1:1,500 (ab40854), all from Abcam, Cambridge, United Kingdom; α-smooth muscle actin (αSMA), 1:500 (Santa Cruz Biotech Inc., Santa Cruz, CA, USA, sc1a4); GAPDH, 1:50,000 (Ambion, ThermoFisher Scientific, Fyrislund, Sweden, AM4300). Horseradish peroxidase-conjugated anti-mouse or anti-rabbit secondary antibodies (ThermoFisher Scientific, Fyrislund, Sweden) were used at 1:20,000 dilution. Triplicate (n_b = 3) biological experiments were performed in 2 technical replicates (n_t = 2) per condition. Densitometric quantification of protein bands was performed using the Fujifilm Intelligent Dark Box II program of a Fuji Aida digital scanner (Fujifilm Nordic AB, Stockholm, Sweden).

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Morén A., Bellomo C., Tsubakihara Y., Kardassis D., Mikulits W., Heldin C.H, & Moustakas A. (2019). LXRα limits TGFβ-dependent hepatocellular carcinoma associated fibroblast differentiation. Oncogenesis, 8(6), 36.

Publication 2019

fibronectin ab41902 ab40854 GAPDH Horseradish peroxidase-conjugated anti-mouse or anti-rabbit secondary antibodies Intelligent Dark Box II

Actin Anti antibodies Antibodies Biological Buffer Cafs Calponin Cells Cold Densitometric Dilution Dish Fatty acid synthase Fibronectin Fuji ii Gapdh Horseradish peroxidase Immunoblot Mouse Phosphate Protein Rabbit Saline Scanner Smad3 Smooth muscle

Corresponding Organization :

Other organizations : Science for Life Laboratory, Uppsala University, University of Crete, Medical University of Vienna, Comprehensive Cancer Center Vienna, Ludwig Cancer Research

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Variable analysis

independent variables

Treatments (unspecified)

dependent variables

Protein expression levels of fibronectin
Protein expression levels of fatty acid synthase (FASN)
Protein expression levels of calponin
Protein expression levels of LXRα
Protein expression levels of Smad3
Protein expression levels of α-smooth muscle actin (αSMA)

control variables

Cell lines: CAFs (2 × 10^4 cells per 12-well dish), Hep3b and SNU423 (5 × 10^5 cells per 60 mm dish), SNU398, SNU449, HLF, Huh7 and PLC/PRF5 (4 × 10^5 cells per 60 mm dish)
Lysis and immunoblot analysis conditions, including buffer pH and temperature
Antibody dilutions used for immunoblotting
Horseradish peroxidase-conjugated secondary antibodies and their dilutions
Number of biological (n_b = 3) and technical (n_t = 2) replicates per condition
Densitometric quantification of protein bands using Fujifilm Intelligent Dark Box II program

controls

No positive or negative controls were explicitly mentioned in the provided information.

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