Conventional molecular biological techniques were used to generate the following expression constructs: N-terminal Myc- and His-tagged human wild-type or mutant UCH-L1; N-terminal S-, GFP-, or GST-tagged human wild-type or mutant parkin. Other expression constructs used in this study include N-terminally HA-tagged Ub-WT, Ub-K48, Ub-K63, Ub-K0 (provided by T. Dawson, Johns Hopkins University, Baltimore, MD) and Ub-K48R and Ub-K63R (provided by M. Wooten, Auburn University, Auburn, AL). Polyclonal anti-UCH-L1 antibody against a synthetic peptide (residues 201-219) of human UCH-L1 was generated in rabbit and affinity-purified as described [40 (link)]. Other antibodies used in this study include anti-actin (clone C4, Millipore), anti-HA (clone 12CA5), anti-Myc (clone 9E10), anti-S-tag (Abcam), anti-ubiquitin (clone P4G7, Abcam), anti-GST (clone B14, Santa Cruz), anti-p62 (BD Biosciences), anti-LC3 (Sigma), and anti-parkin (Cell Signaling). The rabbit polyclonal anti-PINK1 antibody was generated and described in our previous study [41 (link),42 (link)]. Horseradish peroxidase- and fluorophore-conjugated secondary antibodies were from Jackson ImmunoResearch.
Protocol detail
Molecular Cloning and Antibody Generation
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Actin
Anti antibody
Antibodies
Biological
Clone
Horseradish peroxidase
Human
Parkin
Peptide
Rabbit
Ubiquitin
Uch l1
Corresponding Organization :
Other organizations : Emory University
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Variable analysis
independent variables
- N-terminal Myc- and His-tagged human wild-type or mutant UCH-L1
- N-terminal S-, GFP-, or GST-tagged human wild-type or mutant parkin
- N-terminally HA-tagged Ub-WT, Ub-K48, Ub-K63, Ub-K0, Ub-K48R and Ub-K63R
- Not explicitly mentioned
- Polyclonal anti-UCH-L1 antibody against a synthetic peptide (residues 201-219) of human UCH-L1 was generated in rabbit and affinity-purified
- Other antibodies used in this study include anti-actin (clone C4, Millipore), anti-HA (clone 12CA5), anti-Myc (clone 9E10), anti-S-tag (Abcam), anti-ubiquitin (clone P4G7, Abcam), anti-GST (clone B14, Santa Cruz), anti-p62 (BD Biosciences), anti-LC3 (Sigma), and anti-parkin (Cell Signaling)
- The rabbit polyclonal anti-PINK1 antibody was generated and described in our previous study [41, 42]
- Positive control: Not explicitly mentioned
- Negative control: Not explicitly mentioned
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