mRNA Expression Validation by Western Blot

Validation of mRNA (CUL2 and RBBP4) expression at the protein level was performed by western blot. MDMs infected with HIV-1/HIV-2 and uninfected controls, cultured for 7 days post infection were collected by scrapping followed by centrifugation. Proteins were extracted with RIPA buffer (Cat # 89901, Thermo Scientific) supplemented with protease inhibitor cocktail (Cat # 4693159001, Sigma-Aldrich). Equal amount of proteins (5 µg) were used in the Western blot experiment as described earlier^{28 (link)} with m CUL 2 monoclonal antibody (Santa Cruz Biotechnology, Inc., Cat # sc-166506)^{67 (link)}, RBBP4 monoclonal antibody (Santa Cruz Biotechnology, Inc., Cat # sc-373873)^{68 (link)} and β-Actin monoclonal antibody (Santa Cruz Biotechnology, Inc., Cat # sc-47778)^{69 (link)}. ECL western blotting analysis system (Cat # RPN2108, GE Healthcare) was used to visualize the proteins.

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Biswas S., Haleyurgirisetty M., Ragupathy V., Wang X., Lee S., Hewlett I, & Devadas K. (2018). Differentially expressed host long intergenic noncoding RNA and mRNA in HIV-1 and HIV-2 infection. Scientific Reports, 8, 2546.

Publication 2018

RIPA buffer protease inhibitor cocktail β-Actin monoclonal antibody ECL western blotting analysis system

Actin Buffer Centrifugation Hiv 1 Hiv 2 Infection Mdms Monoclonal antibody Mrna Protease inhibitor Proteins Ripa Western blot Western blotting analysis

Corresponding Organization :

Other organizations : Center for Biologics Evaluation and Research

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Variable analysis

independent variables

HIV-1/HIV-2 infection

dependent variables

CUL2 expression at the protein level
RBBP4 expression at the protein level

control variables

Uninfected controls
Protein amount (5 µg) used in Western blot
Culture duration (7 days post-infection)

controls

Positive control: Uninfected controls
Negative control: Not explicitly mentioned

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