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MB Sample ID: SA161886
Local Sample ID: | Mito_011_po |
Subject ID: | SU001794 |
Subject Type: | Cultured cells |
Subject Species: | Homo sapiens |
Taxonomy ID: | 9606 |
Genotype Strain: | HeLa cell line |
Gender: | Not applicable |
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Treatment:
Treatment ID: | TR001807 |
Treatment Summary: | Conditional knockdown and reconstitution of PTPIP51 in HeLa cells were performed as previously reported (Bong et al, 2020). Huma PTPIP51-targeting small hairpin RNA (shRNA) sequences (5’-ATGACTTGATGCCACTATTTA-3’) were inserted into the Tet-pLKO-blasticidin vector. Lentiviruses were produced according to a method described previously (Kim et al, 2018). HeLa cells infected with lentiviruses were selected with blasticidin (10 μg/ml, Invitrogen, USA) for at least 7 days and named HeLa Tet-on-shPTPIP51 cells. The genes encoding human full-length PTPIP51 and PTPIP51_ΔFFAT were cloned into the pCAG-Flag-IRES-puro vector. HeLa Tet-on-shPTPIP51 cells were transfected with pCAG-Flag-IRES-puro empty vector (Mock), PTPIP51, and PTPIP51_ΔFFAT using Lipofectamine 3000 (Life Technologies, USA). Transfected cells were selected with puromycin (2 μg/ml, Amresco, USA) for at least 4 days. For endogenous PTPIP51 knockdown, doxycycline (1 μg/ml, Sigma-Aldrich, USA) was added every two days. Because the shRNAs were designed to target the 3’ UTR of PTPIP51, exogenously added constructs were not targeted. |