Protocol detail

Investigating C9orf72 and TFEB Regulation

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C9orf72-Flag and C9orf72 shRNA constructs were described previously [27 (link)]. GFP-TFEB (38119, Shawn Ferguson, Yale University), pRK5-HA GST RagB (19301, David Sabatini, Whitehead Institute), pRK5-HA GST RagC (19304, David Sabatini, Whitehead Institute), myc-Raptor (1859, David Sabatini, Whitehead Institute), pRK5-HA GST RagB 99L (19303, David Sabatini, Whitehead Institute), and pRK5-HA GST RagC 75N (19305, David Sabatini, Whitehead Institute) were obtained from Addgene.
Antibodies used in the human cell studies include the following: mouse anti-Flag (Sigma), mouse anti-GFP, rabbit anti-GAPDH (ThermoFisher), mouse anti-actin (Santa Cruz), mouse anti-C9orf72 (Bio-Rad), mouse anti-C9orf72 (Proteintech), mouse anti-HA (Bethyl Lab), rabbit anti p-70S6K, rabbit anti p-p70S6K, rabbit anti-RagA, rabbit anti-RagC, rabbit anti-Raptor, rabbit anti-LAMP1, rabbit anti-DYKDDDDK, rabbit anti-pTFEB, rabbit anti-TFEB (Cell Signaling), mouse anti-TFEB (Mybiosources), and rabbit anti-GFP (Abcam).

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Ji Y.J., Ugolino J., Zhang T., Lu J., Kim D, & Wang J. (2020). C9orf72/ALFA-1 controls TFEB/HLH-30-dependent metabolism through dynamic regulation of Rag GTPases. PLoS Genetics, 16(4), e1008738.

Publication 2020

mouse anti-Flag mouse anti-GFP rabbit anti-GAPDH mouse anti-actin rabbit anti-Raptor rabbit anti-LAMP1 rabbit anti-TFEB rabbit anti-GFP

Actin Antibodies Cell Gapdh Human Lamp1 Mouse P70s6k Rabbit Raga Ragc Raptor Shrna Tfeb

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Variable analysis

independent variables

C9orf72-Flag construct
C9orf72 shRNA construct

dependent variables

Not explicitly mentioned

control variables

GFP-TFEB
PRK5-HA GST RagB
PRK5-HA GST RagC
Myc-Raptor
PRK5-HA GST RagB 99L
PRK5-HA GST RagC 75N

controls

Positive control: Not specified
Negative control: Not specified

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