Anti γ tubulin

Cells were lysed in RIPA buffer containing Protease Inhibitor Cocktail kit (Roche, Basel, Switzerland). Then, 20 μg of total protein was separated by SDS-PAGE and then transferred to a PVDF membrane. After blocking in 10% nonfat milk for 1 h at room temperature, membranes were incubated with primary antibodies overnight at 4 °C or 2 h at room temperature. The membranes were then incubated with horseradish peroxidase labeled secondary antibodies and visualized with ECL. The antibodies used and their sources are as follows: HIF-1α (1:500, MA1-516, Thermo Fisher, Waltham, MA, USA), p53Ab1 (1:1000, 9282, Cell Signaling, Beverly, MA, USA), phospho-p53 (Ser15) (1:500, 9284, Cell Signaling), LC-3 (1:5000, 2775, Cell Signaling), PARP (1:1000, 9542, Cell Signaling), caspase-3 (1:1000, 9662, Cell Signaling), GAPDH (1:1000, sc-32233, Santa Cruz, Dallas, TX, USA), Atg7 (1:1000, 8558, Cell Signaling), Atg12 (1:1000,4180, Cell Signaling), HSF1 (1:000, sc-17756, Santa Cruz), HSP70 (1:1000, Stressgen, Farmingdale, NY, USA), P-ser473 AKT (1:1000, 9271, Cell Signaling), AMPK (1:1000, 5831S, Cell Signaling), p-AMPK (1:1000, 5831, Cell Signaling), mTOR (1:1000, A2445, Cell Signaling), p-mTOR (1:1000, 2971, Cell Signaling), p62 (1:1000, 8025, Cell Signaling, Beverly, MA, USA), and anti-γ-tubulin (1:8000, t6557, Millipore, St. Louis, MO, USA).

The following primary antibodies were used in this study: anti-CEP128 [Abcam, ab118797; immunofluorescence (IF) 1:500, western blotting (WB) 1:1000], anti-γ-tubulin (Merck, GTU88; IF 1:1000), anti-p15 (Santa Cruz Biotechnology, sc-271791; WB 1:1000), anti-centrin (Millipore, A302-479A; IF 1:1000), anti-KLC1 (Abcam, ab187179; IF 1:500), anti-HSP90 (BD Biosciences, 610419; WB 1:1000) and anti-β-actin (Santa Cruz Biotechnology, sc-47778; WB 1:1000). The following secondary antibodies were used: anti-mouse IgG Alexa Fluor 488 (Molecular Probes, 1:1000), anti-rabbit IgG Alexa Fluor 555 (Molecular Probes, 1:1000), anti-mouse IgG HRP (Promega, WB 1:10,000) and anti-rabbit IgG HRP (Promega, WB 1:10,000).

For immunoblotting (IB) anti-flagM₂ (IB: 1:10,000, cat#F1804, Merck Millipore, Burlington, MA, USA), anti-GFP (IB: 1:1000, cat#11814460001, Roche, Basel, Switzerland), anti-CNTRB (IB: 1:1000, cat#PA5-96990, Invitrogen, Waltham, MA, USA), anti-R3A (IB: 1:1250, cat#PA566644, Invitrogen, Waltham, MA, USA), anti-R3B (IB: 1:1250, cat#PA551545, Invitrogen), anti-PP4c (IB: 1:1000, cat#MA5-32946, Invitrogen), and anti-αTubulin (IB: 1:10,000, cat#T6199, Merck Millipore, Burlington, MA, USA) were used. For immunostaining, we used anti-γH2AX (IF: 1:500, cat#ab81299, Waltham, MA, USA), anti-γTubulin (IF: 1:300, cat#T6557, Merck Millipore, Burlington, MA, USA) and anti-flag (IF: 1:500, cat#F7425, Merck Millipore, Burlington, MA, USA). Secondary antibodies were the following: goat anti-mouse IgG conjugated to horseradish peroxidase (IB: 1:10,000, cat#P044701-2 Dako, Glostrup, Denmark), donkey anti-rabbit IgG Alexa Fluor 488 (IF: 1:500, cat#A21206, Thermo Fisher Scientific, Waltham, MA, USA) and donkey anti-mouse IgG Alexa Fluor 488 (IF: 1:500, cat#A21202, Thermo Fisher Scientific, Waltham, MA, USA), donkey anti-rabbit IgG Alexa Fluor 594 (IF: 1:500, cat#A21203, Thermo Fisher Scientific, Waltham, MA, USA).