Hsp90 antibody

The NCI/RES-ADR cells were incubated in medium containing various drug formulations for 12 h. After irradiated with laser for 1 min at 1 W cm⁻² (if needed), cells were further cultured for 12 h, washed twice with 1 × PBS at 37 °C, detached using trypsin/ethylenediaminetetraacetic acid (EDTA), and fixed with 4% paraformaldehyde for 20 min at room temperature. After washing, the fixed cells were incubated in 3% BSA and 0.1% TritonX-100 in 1 × PBS at room temperature for 1 h to block potential nonspecific binding and permeabilize the cell plasma membrane, respectively. Following that, the fixed and permeabilized cells were incubated overnight at 4 °C with HSP90 antibody (Cell Signaling, 4874S), HSP70 antibody (Cell Signaling, 4872S), mutant p53 antibody (Abcam, ab32049), HSF-1 antibody (ThermoFisher, PA3–017), and P-gp antibody (Sigma, P7965) at the dilution ratio of 1:200. Unbounded antibody was washed away with 1 × PBS for 3 times. Cells were then incubated with secondary antibody (ThermoFisher) at the dilution ratio of 1:200 in 1 × PBS with 1% BSA at room temperature for 1 h. After washing with 1 × PBS twice, the cells were analyzed using a BD (Franklin Lakes, NJ, USA) LSR-II flow cytometer and Diva software.

Immunoprecipitations were performed using Dynabeads Protein A (Invitrogen) following the manufacturer’s protocol. Briefly, the HSP90 antibody (4877S, Cell Signaling) was preincubated with the beads for 30 min. The bead/antibody mix was then added to 100 μg of whole-cell lysate and incubated for additional 30 min. Samples were washed three times with PBS containing 0.1% Tween-20 and subjected to Western immunoblotting analysis.

We performed western blotting as described previously [39 (link)]. The following primary antibodies were used for protein detection: anti-TET2 (catalog number 61390, Active Motif, Carlsbad, CA), anti-ESRP1 (catalog number GTX131373, GeneTex, Irvine, CA, USA), anti-CD44 (catalog number MAB7045, R&D Systems, Minneapolis, MN, USA), anti-ADARB2 (catalog number sc-73410, Santa Cruz Biotechnology, Dallas, TX, USA), anti-ZEB1 (catalog number 3396, Cell Signaling Technology, Danvers, MA, USA), anti-GRHL2 (catalog number HPA004820, Sigma-Aldrich, St. Louis, MO), and anti-vimentin (catalog number 3932, Cell Signaling Technology). The ECL prime blocking agent (GE Healthcare Life Sciences, Piscataway, NJ, USA) was used for blocking and Lumigen TMA-6 reagents for detection (Lumigen, Inc., Southfield, MI, USA). The blots were re-probed with an HSP90 antibody (catalog number 4875, Cell Signaling Technology) or with an anti-β-actin antibody (catalog number A5316, Sigma-Aldrich) to document equal protein loading. Each western blot was performed at least twice; representative blots are shown. Relative intensities of the bands were determined with ImageJ software (National Institutes of Health, Bethesda, MD, USA).