Western Blotting for Cell Marker Analysis

Western blotting was carried out as previously described (8 (link)). Briefly, mTiD/PLiD lysates were prepared and quantified by using Bio-Rad protein assay kit (Thermo Scientific., Chicago, IL). Cell lysates were subjected to SDS-PAGE and blotted onto PVDF membrane (EMD Millipore, Danvers, MA), blocked and incubated with primary antibodies (mouse anti-E-cadherin (610181, BD Biosciences, San Jose, CA ), mouse anti-vimentin (sc-6260, Santa Cruz, Dallas, TX), rabbit anti-CD31 (PA5–16301, ThermoFisher Scientific, Grand Island, NY), rabbit anti-LYVE1 (ab14917, Abcam, Cambridge, MA), mouse anti-surfactant protein (SP-B, ab3282, or SP-D, ab1778 Abcam, Cambridge, MA), rabbit anti-carcinoembryonic antigen (CEA, ab135781, Abcam, Cambridge, MA), a mouse anti-MUC16 (CA125, ab10029, Abcam, Cambridge, MA) and mouse anti-GAPDH (sc-365062, Santa Cruz, Dallas, TX). Then incubated with appropriate sheep anti-mouse secondary antibodies (NA911V, GE healthcare Life sciences, Pittsburgh, PA) or donkey anti-rabbit (NA934V, GE healthcare Life sciences, Pittsburgh, PA) tagged with HRP. Proteins of interest were detected using enhanced chemiluminescence system (RPN2106, GE healthcare Life sciences, Pittsburgh, PA or 34095, ThermoFisher Scientific, Grand Island, NY) and imaged on chemiDoc™ XRS with Image lab software (Bio-Rad, Hercules, CA).

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Ramamoorthy P., Thomas S.M., Kaushik G., Subramaniam D., Chastain K.M., Dhar A., Tawfik O., Kasi A., Sun W., Ramalingam S., Gunewardena S., Umar S., Mammen J.M., Padhye S.B., Weir S.J., Jensen R.A., Sitta Sittampalam G, & Anant S. (2019). Metastatic Tumor-In-A-Dish, A Novel Multi-Cellular Organoid To Study Lung Colonization and Predict Therapeutic Response. Cancer research, 79(7), 1681-1695.

Publication 2019

PVDF membrane mouse anti-E-cadherin sc-6260 (PA5–16301, ab14917 CA125 sc-365062 NA934V RPN2106 chemiDoc™ XRS Image lab software

Anti antibodies Antibodies Assay Ca125 Cadherin Carcinoembryonic antigen Cell Chemiluminescence Donkey Gapdh Membrane Mouse Proteins Pvdf Rabbit Sds page Sheep Surfactant Vimentin

Corresponding Organization : University of Kansas Medical Center

Other organizations : Children's Mercy Hospital, Savitribai Phule Pune University, National Center for Advancing Translational Sciences, National Institutes of Health

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Variable analysis

independent variables

MTiD/PLiD lysates

dependent variables

E-cadherin
Vimentin
LYVE1
Surfactant protein (SP-B, SP-D)
Carcinoembryonic antigen (CEA)
MUC16

control variables

GAPDH

controls

Positive control: Not explicitly mentioned.
Negative control: Not explicitly mentioned.

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