Protocol detail

Plasmid Transfection Optimization for HeLa, HEK293T Cells

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The following plasmids were used in HeLa-TFEB-GFP cells: pRK5 HA- RAGA (#99710), pRK5-HA GST RAGA 66L (#19300), pRK5 HA- RAGC (#99718), and pRK5-HA GST RAGC 75L (#19305), were purchased from Addgene. The following plasmids were used in HeLa cells: TFEB-GFP wild type, TFEB-GFP S142A, TFEB-GFP S211A, TFEB-GFP S142A/S211A developed by Dr. Shawn Ferguson. The following plasmids were used in HEK293T cells: pEGFP-N1-TFEB (Addgene #38119), pCMV-Tag3B vector plasmid (Agilent) and pCMV-Tag3B expressing myc-FLCN plasmid^{57 (link)}. All plasmids were expressed using Fugene6 reagent (Promega) for 48 h in immunofluorescence experiments and using Lipofectamine 3000 (Invitrogen) for 6 h in immunoblotting experiments.

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Alesi N., Akl E.W., Khabibullin D., Liu H.J., Nidhiry A.S., Garner E.R., Filippakis H., Lam H.C., Shi W., Viswanathan S.R., Morroni M., Ferguson S.M, & Henske E.P. (2021). TSC2 regulates lysosome biogenesis via a non-canonical RAGC and TFEB-dependent mechanism. Nature Communications, 12, 4245.

Publication 2021

pRK5-HA GST RAGA 66L pRK5-HA GST RAGC 75L pEGFP-N1-TFEB Fugene6 reagent Lipofectamine 3000

Cells Flcn Hela cells Immunofluorescence Lipofectamine Plasmids Promega Raga Ragc Tfeb Vector

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Variable analysis

independent variables

PRK5 HA- RAGA (#99710)
PRK5-HA GST RAGA 66L (#19300)
PRK5 HA- RAGC (#99718)
PRK5-HA GST RAGC 75L (#19305)
TFEB-GFP wild type
TFEB-GFP S142A
TFEB-GFP S211A
TFEB-GFP S142A/S211A
PEGFP-N1-TFEB (Addgene #38119)
PCMV-Tag3B vector plasmid (Agilent)
PCMV-Tag3B expressing myc-FLCN plasmid

dependent variables

Not explicitly mentioned

control variables

Kept constant to ensure valid results

controls

No positive or negative controls specified

Annotations

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