The full-length hABHD12 cDNA was cloned into pCMV-Sport6 vector between NotI and SalI restriction sites (GE Life Sciences). The S246A hABHD12 mutant was generated from the same plasmid using Phusion polymerase and DpnI (New England Biolabs) as per manufacturers instructions. Recombinant WT and S246A hABHD12 was generated from HEK293T cells using established transient transfection protocols32 (link). “Mock” control cells were transfected with an empty vector using the same protocol. The cells were harvested by scraping 48 h after transfection, washed with sterile DPBS (3X), resuspended in 1 mL DPBS, and lysed by sonication. The overexpression of hABHD12 was confirmed by gel-based ABPP and western blot analysis on membrane proteomes of the transfected cells, preparations of which are described earlier.
Protocol detail
Overexpression of hABHD12 Mutant in HEK293T Cells
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Abpp
Cdna
Cells
Cells membrane
Plasmid
Proteomes
Sterile
Transfection
Transient
Vector
Western blot analysis
Corresponding Organization :
Other organizations : Indian Institute of Science Education and Research Pune
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Variable analysis
independent variables
- Transfection of hABHD12 wild-type (WT) and S246A mutant
- Overexpression of hABHD12
- Transfection of empty vector (Mock control)
- Positive control: Cells transfected with hABHD12 wild-type (WT)
- Negative control: Cells transfected with empty vector (Mock control)
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