Immunoblotting of Zika Virus Proteins

The Laemmli sample buffer was used in cell lysis to prepare whole cell lysates for SDS-PAGE and WB, as described previously [24 (link),28 (link),29 (link)]. The primary antibodies against Numb (Santa Cruz Biotechnology, Santa Cruz, CA, USA, sc-136554), Myc tag (Santa Cruz Biotechnology, sc-40), GFP (Biolegend, San Diego, CA, USA, 75818-584), ubiquitin (Santa Cruz Biotechnology, sc-8017), GAPDH (Santa Cruz Biotechnology, sc-365062), TUBB1/β-tubulin (Sigma, Livonia, MI, USA, T7816), ZIKV C (GeneTex, Irvine, CA, USA, GTX133317), ZIKV E (B.E.I. Resources, Newport News, VA, USA, NR-50413), ZIKV NS4B (GeneTex, GTX133311), and ZIKV NS5 (GeneTex, GTX133329) were used in this study. Goat anti-mouse or anti-rabbit IgG conjugated with horseradish peroxidase (Bio-Rad, Hercules, CA, USA, 170–5046, and 170–5047) were used as secondary antibodies in this study. For revealing the specific reactions, a chemiluminescence substrate was used, and the signal was recorded digitally using a Bio-Rad ChemiDoc XRS imaging system with the QuantityOne Program, version 4.6 (Bio-Rad Laboratories, CA). Densitometry analysis of the WB images was performed with the QuantityOne Program, version 4.6 (Bio-Rad). All WB images were acquired in the linear range of digital intensity without saturated pixels.

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He J., Yang L., Chang P., Yang S., Wang Y., Lin S., Tang Q, & Zhang Y. (2023). Zika Virus Induces Degradation of the Numb Protein Required through Embryonic Neurogenesis. Viruses, 15(6), 1258.

Publication 2023

Myc tag ubiquitin GAPDH Bio-Rad ChemiDoc XRS imaging system QuantityOne Program, version 4.6

Anti igg Antibodies Cell Chemiluminescence Densitometry Gapdh Goat Horseradish peroxidase Laemmli buffer Linear Mouse Rabbit Sds page Tubulin Ubiquitin Zikv

Corresponding Organization : University of Maryland, College Park

Other organizations : Howard University

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Variable analysis

independent variables

Treatment with primary antibodies against Numb, Myc tag, GFP, ubiquitin, GAPDH, TUBB1/β-tubulin, ZIKV C, ZIKV E, ZIKV NS4B, and ZIKV NS5

dependent variables

Protein expression levels detected by Western blotting

control variables

Cell lysis using Laemmli sample buffer as described previously
Protein detection using chemiluminescence and a Bio-Rad ChemiDoc XRS imaging system
Densitometry analysis of Western blot images using the QuantityOne Program, version 4.6 (Bio-Rad)
Ensuring Western blot images were acquired in the linear range of digital intensity without saturated pixels

controls

Positive controls: Not explicitly mentioned
Negative controls: Not explicitly mentioned

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