Hsc70

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.

Purified Hsc70 was purchased from StressMarq Biosciences (Victoria, Canada). For purification of F-B14, SGTA-F, GFP-F, Hsp105 WT-F, Hsp105 NE*-F and Hsp105 G*-F proteins, confluent HEK 293T cells in 15 cm plates were transfected with pcDNA3.1 (-)-FLAG tag plasmids using 1:4 ratio of DNA to PEI transfection reagent (w/w). After transfection for 24–48 h, cells were washed three times with PBS and harvested using trypsin. Cell pellets were lysed in HNp buffer containing 1% Triton X-100 for 20 min at 4°C. Cell lysates were cleared by centrifugation at 20,000x g for 10 min at 4°C and the supernatant was incubated with 25 μl of anti-FLAG M2 agarose conjugated beads for 2 h at 4°C. Beads were washed three times with HNp buffer and incubated with HKM buffer (20 mM Hepes pH 7.5, 50 mM KCl, 2 mM MgCl₂) containing 0.1% Triton X-100 and 2 mM ATP for 30 min at room temperature. Experiments involving F-B14 contains 0.1% Triton X-100 throughout. Bound proteins were eluted twice using FLAG peptide (100 μl, 0.25 mg/ml; Sigma) for 30 min at 4°C. Eluents were concentrated using centrifugal filters (Amicon Ultra 3K) and the concentrated samples were separated on SDS-PAGE and visualized using Brilliant Blue R250 (Thermo Fisher) staining.