Bladder samples were snap frozen in liquid nitrogen, homogenized with a mortar and pestle, and RNA extracted with the TRIzol (Invitrogen). Quantitative RT-PCR and western blot was performed as previously described39 (link)40 (link). The primers used for RT-PCR is listed below. Antibodies of mouse E-cadherin (20874-1-AP), Vimentin (10366-1-AP), Snail1 (13099-1-AP), Snail2 (Slug, 12129-1-AP), Twist1 (18125-1-AP), Zeb1 (21544-1-AP) and beta-actin (60008-1-Ig) were provided by ProteinTech.
qRT-PCR analysis are:
E-cad F: 5′-CTCCAGTCATAGGGAGCTGTC-3′
E-cad R: 5′-TCTTCTGAGACCTGGGTACAC-3′
Vim F: 5′-TCCACACGCACCTACAGTCT-3′
Vim R: 5′-CCGAGGACCGGGTCACATA-3′
Snail1 F: 5′-CACACGCTGCCTTGTGTCT-3′
Snail1 R: 5′-GGTCAGCAAAAGCACGGTT-3′
Snail2 F: 5′-CAGCGAACTGGACACACACA-3′
Snail2 R: 5′-ATAGGGCTGTATGCTCCCGAG-3′
Twist1 F: 5′-GGACAAGCTGAGCAAGATTC-3′
Twist1 R: 5′-CGGAGAAGGCGTAGCTGAG-3′
Zeb1 F: 5′-ACTGCAAGAAACGGTTTTCCC-3′
Zeb1 R: 5′-GGCGAGGAACACTGAGATGT-3′
Gapdh F: 5′-TGGCCTTCCGTGTTCCTAC-3′
Gapdh R: 5′-GAGTTGCTGTTGAAGTCGCA-3′
qRT-PCR analysis are:
E-cad F: 5′-CTCCAGTCATAGGGAGCTGTC-3′
E-cad R: 5′-TCTTCTGAGACCTGGGTACAC-3′
Vim F: 5′-TCCACACGCACCTACAGTCT-3′
Vim R: 5′-CCGAGGACCGGGTCACATA-3′
Snail1 F: 5′-CACACGCTGCCTTGTGTCT-3′
Snail1 R: 5′-GGTCAGCAAAAGCACGGTT-3′
Snail2 F: 5′-CAGCGAACTGGACACACACA-3′
Snail2 R: 5′-ATAGGGCTGTATGCTCCCGAG-3′
Twist1 F: 5′-GGACAAGCTGAGCAAGATTC-3′
Twist1 R: 5′-CGGAGAAGGCGTAGCTGAG-3′
Zeb1 F: 5′-ACTGCAAGAAACGGTTTTCCC-3′
Zeb1 R: 5′-GGCGAGGAACACTGAGATGT-3′
Gapdh F: 5′-TGGCCTTCCGTGTTCCTAC-3′
Gapdh R: 5′-GAGTTGCTGTTGAAGTCGCA-3′