Bml pw8805 0500

Total proteins were extracted from 4-day-old seedlings or transiently expressed rosette leaves. For western blot analyses, total proteins were extracted from approximately 100 mg of tissue. The following antibodies were used in these experiments: monoclonal anti-GFP (Clontech 632380,1/3000), polyclonal anti-GFP (abcam ab290, 1/2000), anti-FLAG (Abm G191, 1/3000), anti-cMYC (Calbiochem OP10L, 1/3000), anti-ubiquitin P4D1 (Millipore 05-944, 1/2500) and anti-polyubiquitin FK1 (Enzolifesciences BML-PW8805-0500, 1:1000). Uncropped blots are shown in Source Data file. One gram of seedlings or transiently expressed rosette leaves was used for the immunoprecipitation experiments. The tissue was ground in liquid nitrogen, re-suspended in 1 ml of immunoprecipitation buffer (50 mM Tris pH 7.5, 150 mM NaCl, 1% Triton X-100 and protease inhibitor cocktail) and left on a rotating wheel for 30 min at 4 °C. Samples were then centrifuged for 10 min at 20,000 × g at 4 °C. Immunoprecipitations were carried out on 1 mg of total proteins using the EZview^™ Red ANTI-FLAG^® M2 Affinity Gel (Sigma) and GFP-Trap-A (Chromotek) according to the manufacturer’s protocol.

As an indirect measure of protein damage, ubiquitin levels were measured in RBCs,
gill and white muscle using dot blots. Soluble protein (0.5 μg per
sample) was blotted onto a nitrocellulose membrane (BioRad), as well as
0.2 μg of ubiquitin standard for relative quantification (catalogue no.
sc-111402; Santa Cruz Biotechnology, Dallas, TX, USA). The mouse primary antibody
used (1:2500 dilution in 5% BSA/tris-buffered saline with Tween-20;
TBS-T) probed only for polyubiquitinylated proteins and not monoubiquitinylated
proteins or free ubiquitin (BML-PW8805-0500; Enzo Life Sciences, Farmingdale, NY,
USA). The secondary antibody (1:20 000 dilution in 5% BSA/TBS-T)
was a goat anti-mouse IgM (ab97230; Abcam, Cambridge, UK). Blots were visualized as
for HSPs, and the ubiquitin content was quantified relative to the standard run on
each blot.