Anti puromycin 3rh11

Cells were lysed at 4 °C in extraction buffer (0.1 M NaCl, 20 mM HEPES pH 7.5, 1 mM EDTA, 5 mM NaF, 1 mM dithiothreitol, 0.3% Triton X-100, 5% glycerol, 0.25 mM phenylmethylsulfonyl fluoride, and complete protease inhibitor cocktail and phosphatase inhibitors). Homogenates were cleared by centrifugation (12,000 g, 10 min). Coimmunoprecipitation was performed according to previous techniques^{55 (link)}. In vitro IP was performed using purified recombinant proteins instead of the cell lysate. The antibodies used in the study were as follows: Anti-Myc (Cell Signaling, #2276); Anti-DDK (Origene, TA50011); Anti-DJ-1(Santa Cruz, sc27006 and sc32874; Cell Signaling, 2134S); Anti-β-Actin, #4970); Anti-GAPDH (Santa Cruz, sc-32233); Anti-mATP5G1/2/3(abcam, ab180149); Anti-ATPB (abcam, ab14730), and Anti-Bcl-xL(Cell Signaling, #2764); Anti-Puromycin (3RH11) (Kerafast, EQ0001).

The following commercially available antibodies were used in this paper: Anti-HA ab9110 (Abcam), Anti-HA ab18181 (Abcam), Anti-Flag F1804 (Sigma), Anti-Huntingtin Mab2166 (Chemicon, Sigma), Anti-GFP 50430-2-AP (Proteintech), Anti-RFP R10367 (Life Technologies), Anti α-Tubulin 66031–1 (Proteintech), Anti-FAS II-1D4 (DSHB), Anti-Repo 8D12 (DSHB), Anti-Puromycin-3RH11(Kerafast), Anti-Ref2P-ab178440 (Abcam), Anti-HA epitope tag clone 16B12 (Biolegend), Multi Ubiquitin chain monoclonal-cloneFK2 14220 (Cayman), Alexa flour 488 Phalloidin-A12379 (Life Technologies), Alexa flour 555 phalloidin-A34055 (Life Technologies), Alexa Fluor 555 anti-mouse A21424 (Life Technologies), Alexa Fluor 488 anti-mouse A11029 (Life Technologies), Alexa Fluor 555 anti-Rabbit A11034 (Life Technologies), Alexa Fluor 488 anti-Rabbit A21429 (Life Technologies).
Anti-Orb2 and anti-Mrj antibodies were raised against recombinant 6X Histidine-tagged full-length protein, in rabbit and guinea pig for Orb2 and guinea pig for Mrj. For immunoprecipitation, protein-A agarose beads (Repligen 102500–03) and RFP trap magnetic beads (Chromo Tech rtma) were used.