E cadherin 20874 1 ap

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′

Target tissue samples or cells were homogenized in ice-cold RIPA lysis buffer containing 1% protease inhibitor for 30 min. The lysates were centrifuged, and the supernatants were then recovered. The protein concentrations were measured with a bicinchoninic acid protein assay kit. The protein samples were then applied for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The separated proteins were transferred to a polyvinylidene fluoride membrane. The membranes were firstly blocked with 5% non-fat milk and then hybridized with the following antibodies: α-SMA (55135-1-AP; Proteintech, Wuhan, China), collagen I (66761-1-Ig, Proteintech), E-cadherin (20874-1-AP, Proteintech), vimentin (10366-1-AP, Proteintech), OGN (A07061, Boster, Pleasanton, CA, USA), p-mTOR (ab109268, Abcam), mTOR (ab2732, Abcam), Akt (Y409094, ABM, New York, NY, USA), p-Akt (Y011054, ABM) at 4 °C overnight. Horseradish peroxidase-conjugated secondary antibodies and ECL were used for detection and the protein expression was normalized to that of β-actin.

Total cellular protein lysates were collected and analyzed using standard SDS-PAGE and Western blotting protocols. The membranes were blocked using 10% skim milk in TBST for 30 min at room temperature, followed by the incubation with primary antibodies at 4 °C overnight. All antibodies were purchased from Cell Signaling Technology (CST, MA, USA) unless otherwise specified: SREBP1 (ab191857; 1:1,000 dilution), ZEB1 (ab228986; 1:2,000 dilution) from Abcam (Abcam, MA, USA), vimentin (#5741; 1:1000) from Cell Signaling Technology (cell signaling, Danvers, MA, USA), and E-cadherin (20874-1-AP; 1:1,000) and GAPDH (10494-1-AP; 1:10,000) from Proteintech Group (Proteintech, IL, USA), as shown in Supplementary Table S1. Membranes were then washed with TBST and incubated with the secondary antibody, horseradish peroxidase (HRP) conjugate (1:5,000), for 1 h at room temperature, washed with TBST. The immunoreactions were then carried out using an ECL kit (Amersham Biosciences, GE Healthcare, Chicago, IL, USA). GAPDH served as an internal control.

SW480, SW620, HCT116, DLD1, LOVO, HT29, HEK293, and B16 cell lines were obtained from the ATCC and recently been authenticated by STR profiling. The SW480 cell line was cultured in RPMI 1640 (Corning) supplemented with 10% FBS (PAN, P30-3302), and the SW620, HCT116, DLD1, LOVO, HT29, HEK293 as well as B16 cell lines were cultured in DMEM (Corning) supplemented with 10% FBS. These cell lines were cultured in a 37 °C incubator with 5% (v/v) CO₂.
Mouse monoclonal anti-SEC23B antibody was generated against the synthetic peptide LTKPAMPMQQARPAQPQEHP, and was validated in our laboratory (Supplementary Fig. 1a). The commercial antibodies used in this study included GFP (RM1008), GAPDH (RM2002), and α-Tubulin (RM2007) antibodies from Sungene Biotech; EPCAM (66316-1-AP), E-cadherin (20874-1-AP) and PDI (11245-1-AP) from Proteintech; FLAG (M2-3165) from Sigma-Aldrich; GM130 (A5344) from ABclonal; CD9 (sc13118) from Santa Cruz.
The reagents used in this paper included Fibronectin (354008) from Biocoat, Matrigel (356234) from BD, puromycin (sc-205821A) from Santa Cruz, MG132 (Carbobenzoxy-L-leucyl- L-leucyl-L-leucinal) (MB5137) from EPSILON, and chlorhexidine (CHX) (C7698) from SBJBIO, anti-fade mounting reagent (C1210) from Polygen.
C57BL/6N was purchased from Charles River Laboratories.

For immunoprecipitation, antibodies against Flag (sc-166355) and HA (sc-805) were purchased from Santa Cruz. Anti-GST antibody (#2624, Cell Signaling Technology) was used for both immunoprecipitation and immunofluorescence (1:800). For immunoblotting, antibodies against Flag (sc-807, 1:10000; sc-166355, 1:15000), ubiquitin (sc-8017, 1:2000), and PARP-1 (sc-8007, 1:2000) were purchased from Santa Cruz, antibodies against MMP-9 (#10375-2-AP, 1:2000) and E-cadherin (#20874-1-AP, 1:2000) from Proteintech Group, antibodies against HA (#2367, 1:2500) and GAPDH (AP0063, 1:150000) were from Cell Signaling Technology and Bioworld Technology, respectively. The secondary antibody used for immunofluorescence was Rhodamine (TRITC)-conjugated goat anti-mouse immunoglobulin G (1:50, Jackson ImmunoResearch Laboratories).

Radioimmunoprecipitation assay lysis buffer (China Institute of Biotechnology, Beyotime) was added to tissues and SMMC-7721 cells to prepare protein samples. Bicinchoninic acid protein quantification kits (Bio-Rad) were utilized for detection of protein concentration. The lysate was mixed with the loading buffer and denatured in boiling water. Next, the same amount of sample was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to the polyvinylidene fluoride membrane. After blocking the membrane with 5% skim milk at room temperature for 30 min, it was placed with the primary antibody at 4 °C overnight. Next, the membrane and horseradish peroxidase-coupled secondary antibody (ab205718; Abcam) were incubated at room temperature for 1 h. Finally, protein bands were developed using enhanced chemiluminescence Blot substrates (Promega). The antibody information was as follows: PTMA (YN2871, immunoway), E-cadherin (20874-1-AP), N-cadherin (22018-1-AP) (Proteintech), Snail (ab53519), GAPDH (ab8245) (Abcam)^{20 (link)}.