Generation and Characterization of Fluorescent Fusion Constructs

GFP-TRPML1, GFP-TRPML1-7Q, Lamp1-GFP, and Lamp1-mCherry constructs were generated as described previously^{33 (link)}. Dominant-negative Kif5B (human Kif5B a.a. residues 592-963) was subcloned to pmCherry-C1 (Clontech) using Kif5B cDNA as the template. Dominant-negative dynein intermediate chain 2 (GFP-DYNIC2-DN) and GFP-dynamitin (both were gifts from Dr. Kristen Verhey at the University of Michigan) have been characterized previously^{47 (link),67 (link)}. mCherry-ALG2 and GFP-TRPML1-R⁴⁴LK-AAA were provided by Dr. Rosa Puertollano (NHLBI, NIH). mCherry-ALG2-E⁴⁷E¹¹⁴-AA, GFP-TRPML1-R⁴⁴-A, GFP-TRPML1-L⁴⁵-A, Rab7-T²²N-GFP, Rab7-Q⁶⁷L-GFP mutants were generated with a site-directed mutagenesis kit. All constructs were verified with sequencing and confirmed with western blotting.

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Li X., Rydzewski N., Hider A., Zhang X., Yang J., Wang W., Gao Q., Cheng X, & Xu H. (2016). A Molecular Mechanism to Regulate Lysosome Motility for Lysosome Positioning and Tubulation. Nature cell biology, 18(4), 404-417.

Publication 2016

pmCherry-C1

Cdna Dynein intermediate chain Gifts Human Kif5b Lamp1 Rosa Site directed mutagenesis

Corresponding Organization :

Other organizations : University of Michigan–Ann Arbor, Zhejiang University of Technology

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Variable analysis

independent variables

Dominant-negative Kif5B (human Kif5B a.a. residues 592-963)
Dominant-negative dynein intermediate chain 2 (GFP-DYNIC2-DN)
GFP-dynamitin
MCherry-ALG2
GFP-TRPML1-R^44LK-AAA
MCherry-ALG2-E^47E^114-AA
GFP-TRPML1-R^44-A
GFP-TRPML1-L^45-A
Rab7-T^22N-GFP
Rab7-Q^67L-GFP

dependent variables

Not explicitly mentioned

control variables

GFP-TRPML1
GFP-TRPML1-7Q
Lamp1-GFP
Lamp1-mCherry

positive controls

Not specified

negative controls

Not specified

Annotations

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