Phospho p42 p44 mapk

Western blot analysis was carried out as described previously [21 (link)]. In brief, 100 μg of protein from medium or total lysates from HepG2 cells was subjected to Western blotting with monoclonal antibodies against PAI-1 (1:100; American Diagnostics, Pfungstadt, Germany), HIF-1α (1:1000; BD Biosciences, Heidelberg, Germany), phospho p42/p44 MAPK (1:1000), phospho p38 MAPK (1:1000), phospho Akt-T308 (1:1000) and phospho c-Jun-Ser63-Ser73 (1:1000) (Cell Signaling) or rabbit polyclonal antibodies against HO-1 (1:1000; Cell Signaling, Biomol, Hamburg), Golgi membrane (58-kD cis-Golgi protein formiminotransferase cyclodeaminase) (1:10,000; Biosciences, Goettingen, Germany), or α-tubulin (1:10,000; Sigma). The enhanced chemiluminescence (ECL) system was used for detection (Amersham Biosciences).

Protein isolation was acquired with total protein isolation kit (KeyGen, Nanjing, China). The total proteins were then electrophoresed by 10% SDS-PAGE gel and transferred to polyvinylidene fluoride (PVDF, Roche, Switzerland) membranes. The blots were probed or reprobed with antibodies. The membranes were probed using Immobilon Western Chemiluminescent HRP Substrate (Millipore, USA) and autoradiographed. The primary antibodies used were anti-rabbit RBM38, the alia name of RNPC1, (Santa Cruz, USA), PR (Cell Signaling technology, USA), total p42/p44 MAPK (Cell Signaling technology, USA), phospho p42/p44 MAPK (Cell Signaling technology, USA), Wnt (Cell Signaling technology, USA), β-catenin (Cell Signaling technology, USA) anti-mouse β-actin (Cell Signaling technology, USA). The anti-rabbit and anti-mouse secondary antibodies were purchased from Cell Signaling technology (USA). β-actin was used to normalize protein loading. The level of protein was quantified by densitometry.