Protein Purification and Antibody Preparation

A list of plasmids is provided in Table S1. Plasmids were prepared by standard methods. Antibodies against the following proteins were purchased: UBQLN1/2 (Sigma, clone 5F5), UBQLN4 (Abcam ab106443), L9 and Tom20 (Santa Cruz Biotech., T-17 and FL-145), HA (Covance, clone 16B12), Rpt5 (Abcam ab22635), α7 (Enzo Life Sciences PW8110), FLAG (Sigma, clone M2), Hsc/Hsp70 (AssayDesigns, SPA-822), Hsp60 (Abcam ab46798), ClpP (Abcam ab124822), Actin-HRP (Sigma A3854), OxPhos cocktail (Abcam ab110411). FLAG-M2 Affinity resin was from Sigma, and GFP-trap from Chromotek. Antibodies to Bag6, TRC40, SGTA, TRAPα, GFP, and RFP have been described (Fons et al., 2003 (link), Mariappan et al., 2010 (link), Hessa et al., 2011 (link)). Anti-Myc was clone 9E10. Anti-HA used for IPs and blots (e.g., Figure 2A) was raised in rabbits against the KLH-HA peptide conjugate. Recombinant proteins were either purchased (His6-Ubiquitin, E1, and E2 from Boston Biochem) or expressed and purified from E. coli as described (Mateja et al., 2015 (link)).

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Itakura E., Zavodszky E., Shao S., Wohlever M.L., Keenan R.J, & Hegde R.S. (2016). Ubiquilins Chaperone and Triage Mitochondrial Membrane Proteins for Degradation. Molecular Cell, 63(1), 21-33.

Publication 2016

FLAG Hsp60 Actin-HRP OxPhos cocktail FLAG-M2 Affinity resin GFP-trap His6-Ubiquitin

Actin Antibodies Clone Clpp E coli Hsp70 Peptide Plasmids Proteins Rabbits Recombinant proteins Resin Rpt5 Sgta Ubiquitin

Corresponding Organization : MRC Laboratory of Molecular Biology

Other organizations : Chiba University, University of Chicago

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Variable analysis

independent variables

Plasmids used (not listed in the input, but implied based on the description of the plasmid preparation)

dependent variables

Protein expression levels (measured using various antibodies)
Protein-protein interactions (measured using affinity purification)

control variables

Standard methods used for plasmid preparation
Antibodies used for protein detection (e.g., UBQLN1/2, UBQLN4, L9, Tom20, HA, Rpt5, α7, FLAG, Hsc/Hsp70, Hsp60, ClpP, Actin-HRP, OxPhos cocktail)
Affinity resins used for protein purification (e.g., FLAG-M2 Affinity resin, GFP-trap)
Recombinant proteins used (e.g., His6-Ubiquitin, E1, E2)

positive controls

Antibodies against known proteins (e.g., Bag6, TRC40, SGTA, TRAPα, GFP, RFP, Myc, HA)

negative controls

No negative controls explicitly mentioned

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