Snail1

Manufactured by Thermo Fisher Scientific

Sourced in United States

Snail1 is a laboratory equipment product designed for specific research applications. It serves a core function related to the handling and analysis of samples. Further details on the intended use or applications of this product are not available at this time.

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Lab products found in correlation

7 protocols using snail1

Overexpression and Knockdown of EMT Regulators

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Full-length FOXQ1, SNAIL1, TWIST1, ZEB2, and DDR2 plasmids were purchased from Open Biosystems. These genes were subcloned into the pENTR vector (RRID:Addgene_149548) and transferred into a pLenti6 vector (BRID: Addgene_21691) via homologous recombination. The lentivirus for the full-length gene was then generated using the lentivirus-expression system (Invitrogen). In addition, a set of short hairpin RNA (shRNA) clones for DDR2, FOXQ1, or SNAIL1 was purchased from Open Biosystems. The information for the effective shRNA is available in Supplementary Primers and shRNA information. The lentivirus for the shRNA was then generated using the Trans-Lentiviral packaging system (Addgene). The generated lentivirus was then used to infect the targeted model cells. Stable cells were generated after being selected with Blasticidin (10 μg /mL) for the overexpression model or puromycin (12 μg/mL; Invivogen) for the knockdown model.

Mitchell A.V., Wu J., Meng F., Dong L., Block C.J., Song W.M., Zhang B., Li J, & Wu G. (2022). DDR2 Coordinates EMT and Metabolic Reprogramming as a Shared Effector of FOXQ1 and SNAI1. Cancer Research Communications, 2(11), 1388-1403.

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Overexpression and Knockdown of EMT Regulators

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Full-length FOXQ1, SNAIL1, TWIST1, ZEB2, and DDR2 plasmids were
purchased from Open Biosystems (Huntsville, AL). These genes were sub-cloned
into the pENTR vector (RRID:Addgene_149548) and transferred into a pLenti6
vector (BRID: Addgene_21691) via homologous recombination. The lentivirus for
the full-length gene was then generated using the lentivirus-expression system
(Invitrogen, Carlsbad, CA). In addition, a set of shRNA clones for DDR2, FOXQ1,
or SNAIL1 was purchased from Open Biosystems. The information for the effective
shRNA is available in Supplementary Primers and shRNA information. The lentivirus for the
shRNA was then generated using the Trans-Lentiviral packaging system (Addgene,
Watertown, MA). The generated lentivirus was then used to infect the targeted
model cells. Stable cells were generated after being selected with Blasticidin
(10 μg /ml) for the overexpression model or puromycin (12 μg /ml)
(Invivogen, San Diego, CA) for the knockdown model.

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Western Blot Analysis of Epithelial-Mesenchymal Transition

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At the end of experimental time points cells were washed with ice-cold 1X phosphate buffered saline and then lysed using RIPA buffer (Thermo Fisher Scientific). Protein concentrations were determined by BCA kit (Thermo Fisher Scientific). Fifty micrograms of protein lysates were resolved by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred to PVDF membranes. Membranes were blotted using the following antibodies as indicated: SNAIL1 (clone 20C8, 1:500, Thermo Fisher Scientific), USP26 (catalogue # PA5-96893, 1:1000, Thermo Fisher Scientific), E-cadherin (catalogue # 3195, 1:1000; Cell Signaling Technologies, USA)and GAPDH (catalogue # MA5-15738, 1:5000, Thermo Fisher Scientific). GAPDH was used as a loading control. All immunoblot images are representative of 3 experiments.

Li G., Qi H.W., Dong H.G., Bai P., Sun M, & Liu H.Y. (2020). Targeting deubiquitinating enzyme USP26 by microRNA-203 regulates Snail1’s pro-metastatic functions in esophageal cancer. Cancer Cell International, 20, 355.

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Antibody Immunoblotting Protocol

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The antibodies were obtained commercially from various sources – 1) Cell Signaling and Technology -Lamin A/C ( #4777); EpCAM (#2626S); DNMT1 (#5032P); HDAC1 (#5356P); G9a (3306S); Snail1 (3879P); α4-Integrin (#8440S); β-actin (#4970S); Di, Tri-methyl histone H3 (Lys9) (#5327) 2) Thermoscientific – UHRF1 (PA5-27969) 3) BD Biosciences – E-cadherin (610181); p120-catenin (610133); β-catenin (610154) 4) Bethyl laboratories – MARVELD2 (A301-505A) and 5) Sigma/Aldrich – β-tubulin (T0198).

Duvall-Noelle N., Karwandyar A., Richmond A, & Raman D. (2015). LASP-1 – A nuclear hub for the UHRF1-DNMT1-G9a-Snail1 complex. Oncogene, 35(9), 1122-1133.

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Antibody Immunoblotting Protocol

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Duvall-Noelle N., Karwandyar A., Richmond A, & Raman D. (2015). LASP-1 – A nuclear hub for the UHRF1-DNMT1-G9a-Snail1 complex. Oncogene, 35(9), 1122-1133.

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Western Blot Analysis of RPE Proteins

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Lysates were prepared from RPE-choroid preparations from freshly dissected mice (Cryba1^fl/fl, Cryba1 cKO), rat (WT and Nuc1), normal, and AMD RPE cells from human tissues, fetal human RPE cell cultures, and OCM3 cell line. Total protein (50 μg) was loaded for fetal human RPE cell culture and 10 μg protein was loaded for the other samples. Western blots were performed as described previously.^{12 (link),13 (link)} The primary antibodies used in this study were βA3/A1-crystallin (Cat# sc-22398; Santa Cruz Biotechnology and Cat# ab151722; Abcam, Cambridge, MA, USA) as well as a polyclonal antibody developed in our laboratory and previously characterized,^{7 (link)} E-cadherin (Cat# 610181, BD Biosciences, Franklin Lakes, NJ, USA), Vimentin (Cat# 550513; BD Biosciences and Cat# MA5-11883; Thermo Fisher Scientific, Waltham, MA, USA), β-Catenin (Cat# ab16051; Abcam), Cort Actin (Cat# ab33333; Abcam), phospho(Y421)-Cort Actin (Cat# 4569S; Cell Signaling Technology, Danvers, MA, USA), Snail1 (Cat# 14-9859-82; Invitrogen, Carlsbad, CA, USA), and Actin (Cat# A2066; Sigma Aldrich Corp., St. Louis, MO, USA). Secondary antibodies were peroxidase labeled goat anti-rabbit (Cat# 074-1506; KPL, Gaithersburg, MD, USA) and peroxidase labeled goat anti-mouse (Cat# 074-1806; KPL).

Ghosh S., Shang P., Terasaki H., Stepicheva N., Hose S., Yazdankhah M., Weiss J., Sakamoto T., Bhutto I.A., Xia S., Zigler JS J.r., Kannan R., Qian J., Handa J.T, & Sinha D. (2018). A Role for βA3/A1-Crystallin in Type 2 EMT of RPE Cells Occurring in Dry Age-Related Macular Degeneration. Investigative Ophthalmology & Visual Science, 59(4), AMD104-AMD113.

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Western Blot Analysis of EMT Markers

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Cells were lysed using RIPA lysis buffer (Beyotime, Shanghai, China) containing protease and phosphatase inhibitors. Proteins were separated by SDS-PAGE gel and transferred by electrophoresis onto a polyvinylidene fluoride membrane. Subsequently, the membrane was blocked with 5% bovine serum albumin or skim milk in phosphate-buffered saline containing 0.05% Tween-20 followed by hybridization with appropriate primary antibodies at the dilutions recommended by the suppliers and incubated overnight at 4 °C. Next, the membrane was incubated with horseradish peroxidase (HRP)-conjugated secondary antibody, and the bands were visualized using enhanced chemiluminescence substrate (ThermoFisher, 32106). The primary antibodies were the following: PRR15 (RayBiotech, Norcross, GA, USA, 102-22108), PI3K (CST, Boston, MA, USA, 4292S), p-PI3K (Abcam, Cambridge, MA, USA, ab182651), Akt (CST, 4691S), p-Akt (CST, 4060S), mTOR (CST, 2972S), p-mTOR (CST, 2971S), N-cadherin (CST, 13116S), vimentin (Abclonal, Wuhan, China, A2584), Snail1(Invitrogen, Carlsbad, CA, USA, 14-9859-82), and β-actin (CST, 4967) used as a loading control. The intensity of the blots was quantified with software ImageJ and normalized to that of β-actin.

Guo F., Ma J., Li C., Liu S., Wu W., Li C., Wang J., Wang J., Li Z., Zhai J., Sun F., Zhou Y., Guo C., Qian H, & Xu B. (2023). PRR15 deficiency facilitates malignant progression by mediating PI3K/Akt signaling and predicts clinical prognosis in triple-negative rather than non-triple-negative breast cancer. Cell Death & Disease, 14(4), 272.

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