Total p44 42

Transfected PTC cells were lysed in RIPA buffer (Solarbio, China), and phenylmethylsulfonyl chloride was used as a protease inhibitor to stabilize the whole lysate. The extracted proteins were quantified by the bicinchoninic acid assay (Thermo Scientific, United States). Then the proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (BioRad, United States) followed by transferring them to the polyvinylidene difluoride (PVDF) membranes (Millipore, United States). The primary antibodies were as follows: MVP (16478-1-AP, Proteintech), phospho-AKT^Ser473 (4060S, Cell Signaling Technology), total-AKT (4691S, Cell Signaling Technology), phospho-mTOR (381557, Zen Bioscience), total-mTOR (380411, Zen Bioscience), Phospho-p44/42 (4370T, Cell Signaling Technology), total-p44/42 (4695T, Cell Signaling Technology), phospho-p38 (4511T, Cell Signaling Technology), total-p38 (8690T, Cell Signaling Technology), and -Actin (AP0060, Bioworld Technology). Primary antibodies were used for immunoblotting at 1:1,000 dilution. The membranes were then incubated with a secondary antibody (ab97047 or ab6728, Abcam). Eventually, proteins were detected by the chemiluminescence kit (Thermo Scientific), and images of the protein bands were quantified by ImageJ software (NIH, United States).

Lung tissues or cultured cells were homogenized and Western blot was performed as previously described [22 (link)]. Briefly, 20–30 μg of total protein was separated on 10% SDS-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes (Immobilon-P; Millipore Corporation, Billerica, MA). The membranes were then blocked with 5% nonfat milk in PBST (PBS + 0.1% Tween-20) for 20 min and incubated with primary antibody overnight at 4°C. After washing with PBST, the membranes were probed with horseradish peroxidase-conjugated secondary antibody for 2 hrs. Protein bands were visualized with the ECL substrate (Millipore) and scanned using Photoshop. The densitometry unit of the protein expression was normalized to GAPDH levels. The antibodies for HIF-1α, E-cadherin, N-cadherin, vimentin, α-SMA, phospho-p44/42, total p44/42, phospho-p38, and total p38 MAPK were purchased from Cell Signaling Technology (Beverley, MA). Fibronectin and ZEB1 antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). CD44 antibody was purchased from Abcam (Cambridge, MA, USA).