Anti hsc70

Transduced HeLa, NHEK, and serum-starved hTERT-RPE1 cells were washed with PBS and harvested in the FLAG lysis buffer. Supernatants were incubated with 30 μL anti-FLAG-beads (ANTI-FLAG^TM M2 Affinity Gel) overnight at 4 °C and 1 h at RT on a rotator to pool-down ARP-T1-FLAG proteins. Beads were separated by centrifugation for 3 min at 5000 × g, washed five times with TBS, and eluted in 23 μL TBS and 7 μL 5x SDS-sample buffer at 85 °C for 5 min. ARP-T1 precipitation was confirmed using ARP-T1 antiserum (1:2000, GP-SH6), and co-precipitated proteins were analyzed using anti-acetylated tubulin (1:1000, T6793, Sigma-Aldrich), anti-TCP8 / TCP1 theta (1:500, PA5-30403, Thermo Scientific), anti-HSC70 (1:200, PA5-27337, Thermo Scientific), anti-BAG2 (1:100, PA5-30922, Thermo Scientific), anti-gamma tubulin (1:500, ab11316, Abcam), anti-EDH4 (1:1000, Dr. Plomann’s lab), anti-septin 2 (1:2000, HPA018481, Sigma-Aldrich), anti-septin 9 (1:2000, HPA042564, Sigma-Aldrich).

Cells were collected in RIPA lysis buffer (50 mM Tris/HCl pH 7.4, 150 mM NaCl, 1% NP-40, 0, 1% SDS, 1 mM EDTA pH 8, 1 mM PMSF, 0, 5% Sodium Deoxycholate) supplemented with protease inhibitors (Thermo Scientific A32965) and incubated 30 min on ice. After centrifuging at 500 g at 4°C, supernatants were quantified. One milligram (1 mg) of total protein from cell lysate was incubated with 40 μl of Anti-Flag M2 Affinity Gel (Sigma-Aldrich) overnight at 4°C. Then, beads were centrifuged and washed with PBS three times for 10 min at 4°C and then resuspended in 50 μl of resuspension buffer (50 mM Tris/HCl pH 7.4, 5 mM EDTA, 10 mM DTT, 1% SDS) plus 20 μl of SDS 5×. Western blot analysis was performed by using anti-Flag (Sigma-Aldrich, M2), anti-Hsc70 (Thermo Scientific, 13D3) and anti-Lamp2A (Abcam ab18528).