Anti pp2a

Colon tissues (n = 6) were quickly shredded and mixed with protease inhibitor cocktails, which were randomly selected from every group. The mixture was homogenized by ultrasound on crushed ice and centrifuged at 13000g at 4°C for 10 min; the supernatant was extracted; and the protein concentration was determined by BCA protein assay. Equal amounts of total protein (60 μg) were separated by SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membrane using a semidry transfer slot (Bio-Rad, USA). After blocking with 5% nonfat dried milk for 1 h, the membrane was incubated with proportionally diluted antibodies at 4°C overnight. The antibodies included rabbit anti-GAPDH (1:2,000), anti-BCL-2 (1:2,000), anti-BIM-1 (1:1,000), anti-caspase-3 (1:500), anti-BAX (1:1,000), anti-PP2A (1:1,000), anti-β-casein (1:2,500), anti-caveolin-1 (1:1,000), anti-Pim-1 (1:1,000), anti-JAK1 (1:1,000), anti-JAK3 (1:1,000), anti-PIAS1 (1:2,000), anti-PIAS3 (1:2,000), anti-Socs-1 (1:1,000), anti-P-STAT5 (1:1,000), and anti-STAT5 (1:1,000; Abcam). Then, samples were incubated with a corresponding secondary antibody (Abcam) for 1 h. After washing three times with TBST, the blots were visualized by the Proteogel imaging system (FluorChem M, ProteinSimple, USA) and quantified with the Quantity One System (Bio-Rad).

Arecoline hydrobromide purchased from Sigma-Aldrich (St. Louis, MO) used primary antibodies for Western blot: anti-Phospho-Erk1/2 (Thr202/Tyr204) (Millipore, Temecula, CA), anti-Erk1/2 (Cell Signaling Technology, Danvers, MA), anti-Phospho-JNK (Millipore Corp., Billerica, MA), anti-Phospho-IkappaB-alpha (abcam, Cambridge, UK), anti-PP2A (abcam, Cambridge, UK), anti-Phospho-STAT-1 (Millipore Corp., Billerica, MA), anti-ZO-1 (Invitrogen Corporation, Carlsbad, CA) and anti-GAPDH (GeneTex Inc., Hsinchu City, Taiwan). ERK1/2 MAPK inhibitor PD98059 was purchased from Calbiochem (San Diego, CA).

Celastrol and MG-132 were obtained from Calbiochem (Cat #474790; San Diego, CA, USA). PS-341 was obtained from Millennium Pharmaceuticals (Cambridge, MA, USA). Cycloheximide (CHX) was purchased from Beyotime (Jiangsu, China). These reagents were dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich, MO, USA). The primary antibodies were used: anti-CIP2A (HL1925; Santa Cruz Biotechnology, Santa Cruz, CA, USA); anti-PP2A, anti-MCL-1 and anti-Actin (ab137849, ab32087, ab8226; Abcam, Cambridge, MA, UK); anti-GSK3β, anti-BCL-2 and anti-BCL-x_L (#9336, #15071, #2764; Cell Signaling Technology, Danvers, MA, USA). Enhanced chemiluminescence kit was obtained from Yeasen Technology (36222ES60; Shanghai, China).