Bca kit

T24, 5637, RT4, 253J and J82 cells were collected and total protein was collected after lysis and centrifugation. A BCA kit (Thermo Fisher Scientific, Sunnyvale, CA, USA) was used to detect the levels of GP73, E‐cadherin, N‐cadherin and vimentin for screening cell lines. The protein expression levels of GP73, TGF‐β1, Smad2, E‐cadherin, N‐cadherin, vimentin, p‐Smad2 and WT1 in transfected 5637 and 253J cells were determined.
Protein concentrations were measured using a bicinchoninic acid (BCA) kit (Thermo Fisher Scientific). Proteins were separated using 12% sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE), transferred to a polyvinylidene fluoride (PVDF) membrane and then blocked with skimmed milk at room temperature for 1 hr. The samples were incubated overnight with monoclonal antibodies for GAPDH (ab8245), GP73 (ab109628), TGF‐β1 (ab64715), Smad2 (ab76055), p‐Smad2 (ab53100), E‐cadherin (ab33875), vimentin (ab8978) and WT1 (ab201948) (1:300 dilution; Abcam Inc.) at 4°C. After washing three times, the samples were incubated with second antibodies at room temperature for 1 hr. After the membrane was washed for three times, an ECL kit was used to develop Western blotting bands. GAPDH was used as an internal control, and ImageJ2x software Rawak Software, Inc. Germany was used to calculate the relative expression levels of the target proteins.

For phosphorylation of GST-c-JUN TAD, or the ¹⁵N isotope-labeled protein version, 75 μM of substrate was incubated with 290 U recombinant active JNK1 (Proqinase) in 10 mM K₂HPO₄ pH 6.8, 50 mM NaCl, 1 mM ATP, 2 mM DTT, 5 mM MgCl₂ at 20 °C for the indicated times. For phosphorylation of endogenous JNK and c-JUN, cells were treated with 50 μM JNKi for 2 h followed by 15 ng/ml anisomycin treatment for the indicated times at 37 °C. Lysates for SDS-PAGE were prepared in 300 μl RIPA buffer (Thermo Fisher) supplemented with protease inhibitor (Sigma) and phosphatase inhibitor (Cell Signalling). Protein quantification was performed with the BCA kit (Thermo Scientific) and densitometry analysis of the immunoblot with the Image Studio Lite Software (Licor).

After homogenizing the tissues and cells using lysis buffer and centrifugation at 12,000 g for 20 min at 4 °C, protein amounts from all samples were measured with the BCA-kit (Thermo Fisher Scientific; Waltham, MA, USA). Protein samples (50 μg) were loaded onto sodium dodecyl sulfate polyacrylamide gel electrophoresis, and then transferred onto an immobilon-FL transfer membrane (Millipore, Billerica, MA, USA) in a transferring buffer. The membrane was blocked with 5% milk in tris-buffered saline tween-20 (TBST) for 1 h and then incubated overnight at 4 °C with antibodies against TGF-β1, p-smad2, smad2, p-smad3, smad3, smad4, and C-C chemokine receptor (CCR) 2, which were purchased from Cell Signaling Technology (Boston, MA, USA). GAPDH (MB001) was purchased from Bioworld Technology (St Louis Park, MN, USA). The blots were scanned using a two-color infrared imaging system (LI-COR Biosciences: Lincoln, NE, USA). Specific protein expression levels were normalized to GAPDH protein for total cell lysates.