P raf

The operation of the Western blot was referenced the previous instructions [23 (link)]. Briefly, the protein was combined with primary antibodies, and the corresponding secondary antibody was applied to bind to the unique antibody. In the end point, the special protein was visualized via a Visualizer Western Blot Detection Kit (Millipore, Bedford, MA, U.S.A.). The primary antibodies were introduced as follows: HNF4α (ab201460, 1:1000), E-cadherin (ab76055, 1:1000), Vimentin (ab8979, 1:1000), N-cadherin (ab18203, 1:500), phospho-RAF (p-RAF; ab173539, 1:3000), GAPDH (ab8245, 1:5000), RAS (3965, 1:1000), phospho-ERK (p-ERK; 9101, 1:1000), and phospho-MEK (p-MEK; 9154; 1:1000). Of which HNF4α, E-cadherin, Vimentin, N-cadherin, p-RAF, and GAPDH were purchased from Abcam (Cambridge, MA, U.S.A.), RAS, p-ERK, and p-MEK were obtained from Cell Signaling Technology (Boston, MA, U.S.A.).

Immunohistochemistry and immunoblotting were performed as previously described [8 (link)]. Protein concentrations were determined by the BCA Protein Assay Kit (Beyotime, Shanghai, China). Primary antibodies used for immunohistochemistry and/or immunoblotting were as follow: α-SMA, Collagen I, Collagen III, CCN3, TGF-β, p-RAF, p-MEK, p-ERK, E-cadherin, Vimentin and Actin (Abcam, Cambridge, MA, USA). Recombinant human CCN3 was purchased from Peprotech (Rocky Hill, NJ, USA). TGF-β from LX2 was quantified by ELISA (R&D Labs, Minneapolis, MN, USA). Assays were performed according to the manufacturer’s instructions in quadruplicate as previously described [9 (link)].

The treated HCC cells were washed three times with PBS and lysed with RIPA buffer. The protein was obtained and denatured with 5×loading buffer at 100℃ for 10 min, and separated by 10% SDS-PAGE, then transferred to PVDF membranes (Bio-Rad, CA, USA). The membranes were blocked with 5% skimmed milk and then incubated with primary antibodies against UHRF2 (1:1000), p-Raf (1:1000) (Abcam, Burlingame, CA, USA), Ras (1:1000) or Raf (1:1000) (Bioworld Technology, Inc., USA) at 4℃ overnight and then incubated with secondary antibodies (1:5000) at room temperature for 2 h. The bands were detected with Immobilob™ Western Chemiluminescent HRP Substrate (Millipore, Billerica, MA, USA).