pLKO1 non-target control-shRNA (SHC016), FMR1-targeting shRNA (TRCN0000059758) or FXR1-targeting shRNA (TRCN0000159153) constructs were used. We produced lentiviruses via co-transfection of pCMV-d8.91, pVSV-G and pLKO1 into HEK293T cells using Lipofectamine 3000 (Thermo Fisher Scientific, L3000015). Transduction was carried out according to the standard protocol of the ENCODE consortium62 (link). Briefly, viruses were collected from conditioned media after 48 h co-transfection. Lentivirus-containing media was mixed with the same volume of DMEM media that contain polybrene (8 μg/ml), which was used to infect HeLa, SK-N-BE(2), and KELLY cells. After 24 h, cells were incubated with puromycin (2 μg/ml for HeLa and 1 μg/ml for SK-N-BE(2) and KELLY) for 3–7 days. Knockdown efficiency was evaluated by Western blot. Cells were lysed in RIPA containing complete protease inhibitor cocktail. Cell lysates were then resolved through 8% SDS-PAGE and probed by ADAR1 antibody (Santa Cruz, sc-271854), ADAR2 antibody (Santa Cruz, sc-73409), FMRP antibody (Millipore, MAB2160), FXR1P antibody (Bethyl Laboratories, A303–892A), and FXR2 antibody (Sigma-Aldrich, F1554).
Protocol detail
Knockdown of ADAR, FMRP, FXR1P, and FXR2 in Cell Lines
Partial Protocol Preview
This section provides a glimpse into the protocol.
The remaining content is hidden due to licensing restrictions, but the full text is available at the following link:
Access Free Full Text.
Antibody
Cell
Conditioned media
Fmrp
Hela
Lentivirus
Lipofectamine
Polybrene
Protease inhibitor
Puromycin
Ripa
Sds page
Shrna
Transfection
Viruses
Western blot
Corresponding Organization :
Other organizations : University of California, Los Angeles, Center for Autism and Related Disorders, University of California, San Diego, La Jolla Bioengineering Institute, University of California, Davis
Top 5 similar protocols
Variable analysis
independent variables
- PLKO1 non-target control-shRNA (SHC016)
- FMR1-targeting shRNA (TRCN0000059758)
- FXR1-targeting shRNA (TRCN0000159153)
- Knockdown efficiency evaluated by Western blot
- PCMV-d8.91
- PVSV-G
- PLKO1
- Lipofectamine 3000
- Polybrene (8 μg/ml)
- Puromycin (2 μg/ml for HeLa and 1 μg/ml for SK-N-BE(2) and KELLY)
- RIPA buffer with complete protease inhibitor cocktail
- 8% SDS-PAGE
- ADAR1 antibody (Santa Cruz, sc-271854)
- ADAR2 antibody (Santa Cruz, sc-73409)
- FMRP antibody (Millipore, MAB2160)
- FXR1P antibody (Bethyl Laboratories, A303–892A)
- FXR2 antibody (Sigma-Aldrich, F1554)
- None specified
- pLKO1 non-target control-shRNA (SHC016)
Annotations
Based on most similar protocols
Etiam vel ipsum. Morbi facilisis vestibulum nisl. Praesent cursus laoreet felis. Integer adipiscing pretium orci. Nulla facilisi. Quisque posuere bibendum purus. Nulla quam mauris, cursus eget, convallis ac, molestie non, enim. Aliquam congue. Quisque sagittis nonummy sapien. Proin molestie sem vitae urna. Maecenas lorem.
Sign up for free or login to display all annotations
As authors may omit details in methods from publication, our AI will look for missing critical information across the 5 most similar protocols.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!