Monoclonal anti e cadherin

Cells were lysed in RIPA buffer and proteins (30 μg) were separated on 8–12% SDS/PAGE gel and then transferred onto PVDF membranes (Millipore, Billerica, MA). After blocking membranes with non-fat milk for 1 h at room temperature, they were bred with proper dilutions of primary antibodies overnight at 4 °C. The following antibodies were used: monoclonal anti-GAPDH (Catalogue#5174, 1:5000), monoclonal anti-E-cadherin (Catalogue#3195, 1:1000), monoclonal anti-N-cadherin (Catalogue#13116, 1:1000), monoclonal anti-HIF-1α (Catalogue#36196, 1:1000), and monoclonal anti-Vimentin (Catalogue#5741, 1:1000) were all purchased from Cell signaling Technology; polyclonal anti-MMP2 (Catalog number: 10373–2-AP, 1:500) and polyclonal anti-MMP9 (Catalog number: 10375–2-AP, 1:500) were purchased from Proteintech; polyclonal anti-SMAD5 (ab88559, 1:1000) and polyclonal anti-STAT3 (ab119352, 1:1000) were purchased from Abcam; polyclonal anti-TIMP3 was purchased from ABGENT (70R-9697). Next day anti-mouse or anti-rabbit IgG secondary antibody (Cell signaling Technology, USA) were used for 1 h at the concentration of 1:5000 at room temperature and rinsed 10 min by TBST for 3 times. The bands were visualized using an ECL chemiluminescent detection system (Thermo Fisher Scientific, Rochester. NY).

LNCaP and PC3 cells were obtained from the American Type Culture Collection (Manassas, VA) and were routinely cultured in RPMI 1640 (Invitrogen) supplemented with 10% fetal bovine serum (FBS). PC3 stable transfected cells (PC3HO-1 and PC3pcDNA3) were previously described [20 (link)].
Hemin was obtained from SIGMA-Aldrich (UK). For treatments, cells were incubated 24 h in RPMI media containing 10% FBS and then were exposed to Hemin (70 μM, 24 h), unless stated otherwise.
Polyclonal and monoclonal anti-HO-1 antibodies were from Stressgen Biotechnologies Corp. (San Diego, CA). Monoclonal anti-E-cadherin and anti-β-catenin antibodies were from Cell Signaling, Technology (Beverly, MA). Anti-β-Actin antibody was purchased from Sigma (UK). Anti-mouse and anti-rabbit secondary antibodies conjugated with HRP were from Amersham Ltd (UK). Secondary antibodies conjugated with Alexa Fluor 488 or Alexa Fluor 555 were from Molecular Probes, Invitrogen (Grand Island, NY)

The cell protein lysates were mixed with RIPA lysis buffer supplemented with PMSF and protein loading buffer. Then, the lysates were separated by SDS-polyacrylamide gel electrophoresis and transferred to PVDF membrane. The following antibodies, monoclonal anti-GAPDH (Catalogue #5174, 1:1000), monoclonal anti-E-cadherin (Catalogue #3195, 1:1000), monoclonal anti-N-cadherin (Catalogue#13116, 1:1000), monoclonal smad2/3 (Catalogue#8685T, 1:1000) were purchased from Cell signaling Technology; monoclonal anti-EGR1 (Catalogue#55160, 1:1000) was purchased from abcam; monoclonal anti-samd7 (Catalogue#66478-1-lg, 1:1000) was purchased from proteintech; monoclonal smad2/3-phosphorylation (Catalogue#abs130992, 1:1000) were purchased from Absin; anti-mouse and anti-rabbit IgG secondary antibody were purchased from Cell signaling Technology.