Anti gapdh

Cells were centrifuged at 1200 rpm at 4 °C for 10 min to collect cells. Cell pellets were lysed with radioimmunoprecipitation assay (RIPA) buffer (Santa Cruz, Dallas, TX, USA) containing a proteinase inhibitor cocktail (Thermo Fisher Scientific) and incubated for 10 min on ice. The lysed cell pellet was centrifuged at 12,500 rpm at 4 °C for 10 min to isolate the proteins. The concentration of isolated proteins was measured using the bicinchoninic acid protein (BCA) assay. Proteins were then separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidenedifluoride membrane with a 0.2 μm pore size (Bio-Rad, Hercules, CA, USA). The target proteins were detected and analyzed by immunoblotting using the primary antibodies diluted in 0.1% PBS-Tween buffer containing 1% bovine serum albumin and 0.02% sodium azide. These primary antibodies included anti-A20 (1:1000) (Cell Signaling, Danvers, MA, USA) and anti-GAPDH (1:2000) (Enzo Life Science, Farmingdale, NY, USA).

Lysates of cells or mouse tissues (20–100 μg) were subjected to SDS-PAGE and transferred onto a nitrocellulose membrane (GVS Life Science) with a wet blot transfer system (VWR). Membranes were blocked with 2–5% non-fat milk or BSA in TBS-Tween 20 (0.2%) and incubated with primary antibodies with the following dilutions: anti-Hsp90α (1:2000; polyclonal antiserum from Synaptic Systems and monoclonal antibodies from Enzo Lifesciences), anti-Hsp90β (1:2000), anti-Hop (1:1000), anti-GAPDH (1:7500), anti-β-actin (1:5000), anti-Hsp70 (1:2000), anti-Hsc70 (1:2000), anti-c-Raf (1:1000), anti-Ub (1:5000), anti-p23 (1:1000), anti-Cdc37 (1:1000), anti-Akt (1:1000), anti-Hsf1 (1:1000), anti-Hsp40/Hdj1 (1:1000), anti-Hsp110 (1:1000), anti-Aha1 (1:2000), anti-Hsp25/27 (1:2000), anti-Puromycin (1:22,000), anti-p-mTOR (1:1000), anti-mTOR (1:2000), anti-p-S6 (1:1000), anti-p-eIF2α (1:1000), anti-eIF2α (1:1000). Membranes were washed with TBS-Tween 20 (0.2%) and incubated with the corresponding secondary antibodies: anti-mouse IgG-HRP (1:10,000), anti-rabbit IgG-HRP (1:10,000), and anti-rat IgG-HRP (1:10,000). Immunoblots were developed using the WesternBright^TM chemiluminescent substrate (Advansta). Images were recorded by using a LI-COR Odyssey or Amersham ImageQuant 800 image recorder.

Cholera toxin (100) and insulin (16634) were from List Biological (Campbell, CA, USA) and Invitrogen (Carlsbad, CA, USA), respectively. Polyethyleneimine (PEI; 25 kDa linear (23966) was from Polysciences (Warrington, PA). Chelex 100 resin was purchased from Biorad (142-2832; Mississauga, ON, Canada). All other reagents were purchased from Sigma (St. Louis, MO, USA). The antibodies used were: anti-V5 (R960-25; Invitrogen, Carlsbad, CA, or ab9113, Abcam, Cambridge, MA), and anti-E2F1 (TA308764; OriGene, Rockville, MD). Antibodies against cyclobutane pyrimidine dimers (CPD) were from CosmoBio Co. (NMDND001, clone TMD-2, Tokyo, Japan); anti-double strand (ds) DNA (ab27156, Abcam, Cambridge, MA); anti-HA (Y-11) and anti-Rad23A (D-6) antibodies were purchased from Santa Cruz (Santa Cruz, CA), anti-GAPDH was from Enzo Life Sciences (ADI CSA 335, Farmingdale, NY); anti-FLAG M2 (F1804) and anti-γ-tubulin (T6557) were from Sigma (St. Louis, MO). Horseradish peroxidase-conjugated goat anti-mouse (115–035-146) and anti-rabbit (111-035-144) antibodies were from Jackson Immuno Research Laboratories (West Grove, PA). Alexa Fluor®-conjugated phalloidin (A12379), goat anti-rabbit and goat anti-mouse IgG were from Molecular Probes/Invitrogen (Eugene, OR).