Human LRG1 (NM_052972) carrying a 6xHis tag expression vector, pcDNA3.1-LRG1, was generated as previously described (11 (link)). rhLRG1 protein was expressed in Freestyle 293 T cells (Invitrogen) and purified as previously described (11 (link)). siRNA oligonucleotides (cat. no. L-015179-01; Dharmacon) were used for LRG1 gene knockdown, while control siRNA (D-001810-10-20; Dharmacon) was used as a negative control. Transfection was performed using Lipofectamine 2000 (Invitrogen) for HaCaT cells and RNAiMAX (Invitrogen) for dHL-60 cells according to the manufacturer’s protocol.
Protocol detail
Recombinant LRG1 Protein Expression and Knockdown
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293 t cells
Cells
Gene knockdown
Hacat cells
Human
Lipofectamine 2000
Oligonucleotides
Protein
Sirna
Transfection
Vector
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Variable analysis
independent variables
- Expression vector pcDNA3.1-LRG1 with 6xHis tag
- SiRNA oligonucleotides (cat. no. L-015179-01) for LRG1 gene knockdown
- Control siRNA (D-001810-10-20)
- Not explicitly mentioned
- Freestyle 293 T cells (Invitrogen) used for protein expression
- Purification of rhLRG1 protein as previously described
- Lipofectamine 2000 (Invitrogen) used for HaCaT cell transfection
- RNAiMAX (Invitrogen) used for dHL-60 cell transfection
- Positive control: Not explicitly mentioned
- Negative control: Control siRNA (D-001810-10-20)
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