Anti hbp1

Cells were transfected with 40 nM siHBP1 and siNC for 24 h and then seeded in 6-well plates as described above. After 24 h culture, cells were fixed with 4% paraformaldehyde solution and followed by the protocols as previously described^{41 (link)}. The photographs were taken at five different fields at 20× under BX60 fluorescence microscope (Olympus, Japan) and were scored for HBP1 expression. The anti-HBP1 (Millipore, Billerica, MA,USA, 1:200) was a rabbit-IgG antibody and the Integrated Optical Density (IOD) and area were measured by Image-Pro Plus software (MediaCybernetics, USA), IOD/area was set as a semi-quantitative index for the expression of HBP1 protein.

HK1, HNE1, and CNE2 cell lines were transfected as described above. After 48 h transfection, proteins were extracted in the defined volume of RIPA lysis buffer containing 1.0 mM PMSF, protease inhibitor cocktail and DTT, and 50 μg proteins was loaded in SDS-PAGE gel electrophoresis^{10 (link)}. The proteins were transferred to PVDF Membrane (Milipore, Darmstadt, Germany) which were incubated with following primary antibodies: anti-HBP1 (Milipore, Darmstadt, Germany), anti-ZO-1, anti-E-cadherin, anti-N-cadherin, anti-Vimentin, anti-β-catenin, anit-MMP9, anti-NF-κB, anti-caspase 3 and cleaved caspase 3, anti-caspase 7, anti-PARP and cleaved PARP, anti-caspase 9 and cleaved caspase 9, anti-p18^INK4C (p18), anti-p21^Waft/Cip1 (p21), anti-p27^Kip1 (p27), anti-Cyclin D1, anti-Cyclin D3, anti-CDK2, anti-CDK4, anti-CDK6 (Cell Signaling Technology, Danvers MA, USA), and β-actin (ABclonal, Cambridge, MA, USA) as an internal reference. Then the PVDF Membranes were incubated with HRP-linked anti-Rabbit or anti-Mouse IgG antibody according to the isotypes of the primary antibody. The membrane was imaged using ChemiDoc MP System (Bio-Rad, Hercules, CA, USA) and the images were analyzed using Image Lab Software (Bio-Rad, CA, USA).

The paraffin sections staining and evaluation were performed as previously described^{10 (link)}. The dilution ratio of anti-HBP1 (Millipore, Billerica, MA, USA) and Ki67 (BBI Life Sciences, Shanghai, China) was 1: 200 and 1: 300, respectively. All sections were scored independently by three researchers according to the clinical data and clinicopathological features. On the high magnification, the comprehensive staining intensity and the proportion of positive cells was used for semi-quantitative determination. Dying color was scored as 0 for no staining, 1 for light yellow, 2 for light brown and 3 for brown. Proportion of positive cells was graded as score 0, <5%; score 1, 5–25%; score 2 points, 26–50%; score 3, 51–75%; and score 4, >75%. Two scores multiplied together and the total score divided into four ranks: 0 for negative expression (−), 1 to 4 for weak expression (+), 5 to 8 for middle positive expression (+ +), and 9 to 12 for strong positive expression (+ + +).