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3 protocols using cldn1

1

Western Blot Characterization of Protein Markers

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Protein lysates were prepared with RIPA Buffer (PI-89900, Thermo Fisher Scientific) and quantified using BCA Protein Assay Kit (23225, Pierce Biotechnology). Equal amounts of total protein were loaded for immunoblot analysis. Immunoblots were probed with antibodies against MYC (5605S, Cell Signaling Technology), SMAD4 (38454S, Cell Signaling Technology), GLI3 (sc-74478, Santa Cruz Biotechnology), MYBL2 (sc-390198, Santa Cruz Biotechnology), LOXL2 (ab96233, Abcam), CLDN1 (4933S, Cell Signaling Technology), HGK (3485, Cell Signaling Technology), NRAS (ab77392, Abcam), E-cadherin (ab40772, Abcam), Vimentin (D21H3, Cell Signaling), LAMB1 (D4Q4Z, Cell Signaling) and GAPDH (MA5–15738, Thermo Fisher Scientific) overnight at 4°C followed by incubation for 1 hr at room temperature with corresponding HRP conjugated goat anti-mouse (31430, Thermo Fisher Scientific) or goat anti-rabbit (31460, Thermo Fisher Scientific) secondary antibodies. Western Blots were then developed using ECL detection kit (34096, Thermo Fisher Scientific) and captured on an Amersham Imager 600 (GE Healthcare). Densitometry analysis was performed using Image J software to quantify each protein band, and were normalized against LAMB1 for nuclear fraction or GAPDH.
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2

Comprehensive Protein Analysis of EMT

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Total protein was isolated from cells using RIPA lysis buffer (Beyotime). Western blot analysis was performed as previously described 15 (link). The intensity of the protein bands was quantified using ImageJ software (NIH). Primary antibody against FN1 (1:1000, ab32419) was purchased from Abcam (Shanghai, China). Antibodies against THBS1 (1:1000, 18304-1-AP), CPED1 (1:1000, 20924-1-AP), AMOTL2 (1:1000, 23351-1-AP), TGFβ1 (1:1000, 21898-1-AP), TGFβ2 (1:1000, 19999-1-AP), and GAPDH (1:10000, 60004-1-Ig) were purchased from Proteintech Group (IL, USA). Antibodies against E-cadherin (1:500, #3195), N-cadherin (1:500, #13116), Slug (1:1000, #9585), Snail (1:1000, #3879), ZO1 (1:1000, #8193), β-catenin (1:1000, #8480), CLDN1 (1:1000, #13255), MMP2 (1:1000, #40994), and MMP9 (1:1000, #13667) were purchased from Cell Signaling Technology (MA, USA).
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3

Comprehensive Antibody Panel for Cell Signaling

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Those obtained from Cell Signaling Technology included CCND1 (E3P5S XP), HRAS (D2H12), VIM (D21H3 XP), CLDN1 (D5H1D XP), β-CATENIN (D10A8 XP), SNAIL (C15D3), CDH1 (24E10), ZEB1 (E2G6Y XP), RELA (p65) (D14E12 XP), STING (D1V5L), TBK1 (D1B4), IRF3 (D83B9), RELB (C1E4), IKBA (44D4), NF-KB2 (p100/p52) (4882), H3 (D1H2 XP). Other antibodies included HEY1 (ProteinTech 19929–1-AP), ITGA6 (BD Horizon GoH3, BV650), ITGB1 (BioLegend HMB1–1 APC/Cy7).
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