Molecular Tools for Autophagy Research

HA-NEDD4^WT (Addgene, 27002), and HA-NEDD4^C867A (Addgene, 26999) were gifts from Dr. Joan Massague.⁵⁸ (link) MYC-ULK1^WT (Addgene, 27629), MYC-ULK1^K46I (Addgene, 27630), and MYC-ULK1^S4A (Addgene, 27631) were gifts from Dr. Reuben Shaw.⁵⁹ (link) FLAG-ubiquitin WT and related mutants (K6WT, K11WT K27WT, K29WT, K33WT, K48WT, K63WT and KallR) were provided by the MRC Protein Phosphorylation and Ubiquitylation Unit, UK. YFP-Atg8-family proteins encoding plasmids were a kind gift from Dr. Felix Randow (MRC Laboratory of Molecular Biology, UK). MYC-BECN1 plasmid (Biocat GmbH, MR207162-OR), MG132 (Merck Chemicals GmbH, 474791), Torin1 (Tocris Bioscience, 4247), cyclohexamide (Sigma-Aldrich, C1988) and choloroquine (Sigma-Aldrich, C6628) were purchased as indicated. Antibodies used in this study include: anti-ACTB (Sigma-Aldrich, A2228), anti-LC3 (MBL International, PM036), anti-human NEDD4 (Cell Signaling Technology, 3607), anti-mouse NEDD4 (BD Biosciences, 611481), anti-K48 ubiquitin (Merck Millipore, 05–1037), anti-K63 ubiquitin (Enzo Life Sciences, HWA4C4), anti-ubiquitin (FK2) (Enzo Life Sciences, BML-PW8810), rabbit anti-BECN1 (Cell Signaling Technology, 3495), mouse anti-BECN1 (Cell Signaling Technology, 4122), anti-ULK1 (Cell Signaling Technology, 8054), anti-LC3B (Sigma-Aldrich, L7543), anti-LAMP2 (Santa Cruz Biotechnology, SC-18822).

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Pei G., Buijze H., Liu H., Moura-Alves P., Goosmann C., Brinkmann V., Kawabe H., Dorhoi A, & Kaufmann S.H. (2017). The E3 ubiquitin ligase NEDD4 enhances killing of membrane-perturbing intracellular bacteria by promoting autophagy. Autophagy, 13(12), 2041-2055.

Publication 2017

MG132 Torin1 cyclohexamide choloroquine anti-ACTB anti-LC3 anti-ubiquitin (FK2) rabbit anti-BECN1 mouse anti-BECN1 anti-ULK1 anti-LC3B anti-LAMP2

Antibodies Atg8 family proteins Becn1 Gifts Human Lamp2 Mg132 Mouse Phosphorylation Plasmid Protein Rabbit Ubiquitin Ubiquitylation Ulk1

Corresponding Organization : Max Planck Institute for Infection Biology

Other organizations : Shanghai Pulmonary Hospital, Tongji University, Max Planck Institute of Experimental Medicine

Top 5 similar protocols

Variable analysis

independent variables

HA-NEDD4^WT (Addgene, 27002)
HA-NEDD4^C867A (Addgene, 26999)
MYC-ULK1^WT (Addgene, 27629)
MYC-ULK1^K46I (Addgene, 27630)
MYC-ULK1^S4A (Addgene, 27631)
FLAG-ubiquitin WT and related mutants (K6WT, K11WT K27WT, K29WT, K33WT, K48WT, K63WT and KallR)
YFP-Atg8-family proteins encoding plasmids
MYC-BECN1 plasmid (Biocat GmbH, MR207162-OR)
MG132 (Merck Chemicals GmbH, 474791)
Torin1 (Tocris Bioscience, 4247)
Cyclohexamide (Sigma-Aldrich, C1988)
Choloroquine (Sigma-Aldrich, C6628)

dependent variables

Not explicitly mentioned

control variables

Not explicitly mentioned

controls

Positive controls: Not specified
Negative controls: Not specified

Annotations

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