Western Blotting Protein Detection Protocol

Western blots were performed as previously described (49 (link)). In brief, protein lysates from cell culture or tumor tissues were extracted in ice-cold RIPA buffer (50 mM Tris-Cl, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) containing cocktails of protease (Thermo Scientific) and phosphatase inhibitors (Sigma-Aldrich), by centrifugation (13000g, 15 min) at 4°C following 3 freeze-thaw cycles. Proteins were separated by SDS-PAGE and transferred to methanol activated polyvinylidene difluoride (PVDF) membrane (VWR). The primary antibodies used include the following: rabbit anti-mouse Fbln5 (1:1000), rabbit anti-human Fbln5 (1:500) (HPA000868, Sigma Aldrich), anti-Nqo1 (1:1000) (ab34173, Abcam), anti-α-Tubulin (1:1000) (ab4047, Abcam) and anti-β-actin (1:5000) (A2066, Sigma-Aldrich). HRP-conjugated donkey anti-rabbit IgG (1:10000) (Jackson Immunoresearch) as secondary antibodies were used.

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Wang M., Topalovski M., Toombs J.E., Wright C.M., Moore Z.R., Boothman D.A., Yanagisawa H., Wang H., Witkiewicz A., Castrillon D.H, & Brekken R.A. (2015). Fibulin-5 blocks microenvironmental ROS in pancreatic cancer. Cancer research, 75(23), 5058-5069.

Publication 2015

cocktails of protease and phosphatase inhibitors PVDF) membrane ab34173 ab4047 A2066 HRP-conjugated donkey anti-rabbit IgG

Actin Anti igg Antibodies Buffer Cell culture Centrifugation Cold Donkey Freeze Human Inhibitors Membrane Methanol Mouse Nacl Np 40 Nqo1 Phosphatase Polyvinylidene difluoride Protease Proteins Rabbit Ripa Sds page Sodium deoxycholate Tissues Tris Tumor Western blots α tubulin

Corresponding Organization : The University of Texas Southwestern Medical Center

Other organizations : The University of Texas MD Anderson Cancer Center

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Variable analysis

independent variables

None explicitly mentioned

dependent variables

Protein expression levels of Fbln5, Nqo1, α-Tubulin, and β-Actin

control variables

Protein extraction in ice-cold RIPA buffer containing protease and phosphatase inhibitors
Centrifugation of protein lysates at 13000g for 15 min at 4°C following 3 freeze-thaw cycles
SDS-PAGE separation of proteins and transfer to PVDF membrane
Use of primary antibodies against Fbln5, Nqo1, α-Tubulin, and β-Actin
Use of HRP-conjugated secondary antibody (donkey anti-rabbit IgG)

positive controls

None explicitly mentioned

negative controls

None explicitly mentioned

Annotations

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