Generation of shRNA and HA-tagged Mutant Constructs

shRNA against μ2 and HA-μ2 plasmids resistant to shRNA μ2 were constructed as previously described24 (link). 60-mers oligonucleotides containing rat μ2 subunit cDNA target sequences (GTGGATGCCTTTCGCGTCA) of AP-2 were synthesized, annealed, and ligated into pSUPER vector according to manufacturer’s instruction. HA-μ2 construct was generated using Quick Change Site-Directed mutagenesis (Stratagene). The target sequence for shRNA μ2 was mutated to GTGGACGCTTTCCGGGTCA without the amino acid sequence change. EGFP-C2 is from clontech, GFP-Tau, VMAP2-GFP, GFP-synapsin-1, Na_v1.6-GFP was cloned as previously described52 (link). GCaMP6f from Addgene30 (link) is conjugated to synaptophysin.

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Kyung J.W., Cho I.H., Lee S., Song W.K., Ryan T.A., Hoppa M.B, & Kim S.H. (2017). Adaptor Protein 2 (AP-2) complex is essential for functional axogenesis in hippocampal neurons. Scientific Reports, 7, 41620.

Publication 2017

EGFP-C2 GCaMP6f

Amino acid sequence Cdna Mers Oligonucleotides Plasmids Shrna Site directed mutagenesis Subunit Synapsin 1 Synaptophysin Vector

Corresponding Organization : Kyung Hee University

Other organizations : Dartmouth College, Scripps Korea Antibody Institute, Gwangju Institute of Science and Technology, Cornell University

Top 5 similar protocols

Variable analysis

independent variables

ShRNA against μ2
HA-μ2 plasmids resistant to shRNA μ2

dependent variables

Cellular processes or functions affected by the knockdown of μ2 subunit and the expression of HA-μ2 plasmids

control variables

EGFP-C2
GFP-Tau
VMAP2-GFP
GFP-synapsin-1
Na^1.6-GFP
GCaMP6f conjugated to synaptophysin

controls

Positive control: HA-μ2 plasmids resistant to shRNA μ2
Negative control: EGFP-C2, GFP-Tau, VMAP2-GFP, GFP-synapsin-1, Na^1.6-GFP, GCaMP6f conjugated to synaptophysin

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