Rabbit monoclonal anti phospho p44 42mapk erk1 2

Cells were precipitated with 6% ice-cold TCA for an hour and then centrifuged 10 min at 9000 rpm. Total cellular protein pellets were resuspended in electrophoresis sample buffer (62.5 mM Tris–HCl, pH 6.8, 2% SDS, 10% glycerol, 5 mM EDTA, 125 mg/mL urea, 100 mM dithiothreitol) and equal amounts of protein were loaded on 10% acrylamide gels. The following primary antibodies were used: rabbit monoclonal anti-phospho-p44/42MAPK (ERK1/2) (Cell Signaling, 4370S, dilution 1:2000), mouse monoclonal anti-ERK1/2 (Cell Signaling, 4696S, dilution 1:2000), rabbit polyclonal anti-beta-tubulin (Abcam, ab6046, 1:2000),, rabbit polyclonal anti-PD-L1 (Cell Signaling, 13,684 T, 1:1000). Then HRP-conjugated anti-rabbit and anti-mouse secondary antibodies (Jackson ImmunoResearch, dilution 1:10000) were applied and for detection Pierce ECL Western Blotting Substrate (Thermo Scientific) and luminography was used.

After proteins were extracted and assessed by protein assay as previously described [7 (link)], samples containing 40 μg of protein were subjected to 10% SDS gel electrophoresis, followed by Western blotting using the following primary antibodies:

Rabbit monoclonal anti E-cadherin (1:1000 dilution; Cell Signaling Technology, Inc., USA),

Rabbit monoclonal anti-α-SMA (1:1000 dilution; Cell Signaling Technology, Inc., USA),

Rabbit monoclonal anti-phospho-Smad2/Smad3 (1:1000 dilution; Cell Signaling Technology, Inc., USA),

Rabbit monoclonal anti-Smad2/3 (1:1000 dilution; Cell Signaling Technology, Inc., USA),

Rabbit monoclonal anti-phospho-p44/42 MAPK (Erk1/2) (1:2000 dilution; Cell Signaling Technology, Inc., USA),

Rabbit monoclonal anti-p44/42 MAPK (ERK1/2) (1:2000 dilution; Cell Signaling Technology, Inc., USA), and

Anti-β-actin (1:4000 dilution; Proteintech Group, Inc., USA).

After washing with primary antibodies, the membranes were incubated with secondary antibodies (1:20,000; Abcam, Cambridge, MA, USA). An enhanced chemiluminescence (ECL) Western blotting kit (Applygen Technologies, Inc., Beijing, China) was used to assess target band expression. β-Actin was used as the internal control, and the relative expression levels of E-cadherin, α-SMA, p-SMAD2/3/SMAD2/3 and phospho-ERK1/2/ERK1/2 in each experimental group were calculated.