Hsc70

The following antibodies were used for Western Blot and co-immunoprecipitation: p53 (DO-1 and CM-1) and MDM2 (4B2) were a kind gift from B.Vojtesek, p63 (4A4, Santa Cruz Biotechnology), p73 (5B429 mouse monoclonal, Abcam; rabbit polyclonal – gift from B.Vojtesek), HSP70 (SPA-812, Stressgen; 6B3, Cell Signaling), HSP40 (SPA-400, Stressgen), HSP90 (SPA-835, Stressgen), HSC70 (SPA-815, Stressgen), HA (3F10, Roche Molecular Biochemicals; 12CA5, Abcam), PARP (9542S, Cell Signaling), β-actin-HRP (AC-15, Sigma-Aldrich). Secondary antibodies used in Western blot were conjugated with HRP (Calbiochem).

Antibodies (dilutions are indicated in brackets for western blot (WB), immunofluorescence (IF), or immunoprecipitation (IP)) against FLAG (Sigma, clone M2; Sigma, produced in Rabbit, IP 3 μl/sample, IF 1:100, WB 1:1000), FLAG (Sigma, clone M2; Sigma, M, Wb 1:1000, IF 1:200), ubiquityl-histone H2A (Millipore, clone E6C5), ubiquitin (Norvus Biologicals, FK2, M, WB 1:1000, IF 1:1000; Dako WB), K48-linkage specific polyubiquitin (Enzo lifesciences, WB 1:1000), K63-linkage specific polyubiquitin (Cell Signaling, clone D7A11, 1:1000), myc (MBL, clone PL14, WB 1:3000, IF 1:100), HSC70 (Stressgen, WB 1:5000, IF 1:100), LC3B (Novus Biologicals, NB100-220), GFP (clonetech, 632381), p62 (Enzo Life Sciences, BML-PW9860), Lamin A/C (Santa Cruz, 4A58), HSC70 (Stressmarq biosciences), HSP70 (Stressgen, clone SPA-810, WB 1:1000, IF 1:50), HSPA1A (Enzo life sciences, Rb, WB 1:1000), HSPB1 (Stressmarq biosciences), GAPDH (Fitzgerald, clone 6C5, WB 1:50,000), histone H2A (Abcam, WB 1:5000), MYC (Clonetech, Mountain View, CA, USA), and DNAJB1/Hsp40 (Stressgen, San Diego, CA, USA, Rb, 1:1000) were used.
MG132 (20 µM for 3–6 h), rapamycin, Pepstatin A (10 μg/ml), E64d (10 μg/ml), 3-methyladenine (3-MA, 10 mM) ammonium chloride (NH₄Cl, 20 mM) were from sigma.

These experiments were performed as described in [37 (link)]. Briefly, seedlings were homogenized in extraction buffer (50 mM Tris pH 7.5, 150 mM NaCl, 1 mM EDTA, 10% glycerol, 0.1% Triton X-100, 5 mM DTT, 1% protease inhibitor cocktail (Sigma-Aldrich), 10 μM MG132, phosSTOP phosphatase inhibitor cocktail (Roche, 1 tablet/5 ml buffer)). After centrifugation, 1.5–3 mg of total protein of the supernatant was subjected to co-immunoprecipitation using the magnetic μMACS Anti-HA isolation kit (Miltenyi Biotec) for HA-tagged bait proteins and magnetic μMACS Anti-GFP isolation kit (Miltenyi Biotec) for YFP-tagged bait proteins according to the manufacturer´s instructions. Twice as much total protein was added for YFP-COP1 spaQn seedlings than for YFP-COP1 seedlings. Proteins were detected by Western blot using antibodies against GFP (Roche Diagnostics, Mannheim, Germany), HA (Roche Diagnostics, Mannheim, Germany), HSC70 (Stressgen Biotechnologies, San Diego, USA), α-tubulin (Sigma-Aldrich, Munich, Germany), CRY1 [40 (link)] and CRY2 [41 (link)]. All experiments were repeated 3–6 times with similar results, except for the experiments shown in Fig 4A and S1 Fig which were repeated twice with similar results. A representative image is shown for each experiment.