Ab53497

HGFs cells were planted on the plate and treated with drugs. The cells were fixed with 4% paraformaldehyde, and permeabilized with Triton X-100. PBS was rinsed and sealed with 5% BSA. Then anti-HSP90α (abcam, ab79849, 1:100), anti-HSP90β (abcam, ab53497, 1:100) antibody were added and incubated at 37 °C for 3 h. After washing with PBS, the secondary antibody IgG H & L (abcam, ab150080, 1:500) was incubated for 30 min. DAPI was added dropwise to dye the nucleus under dark conditions, and sealed slices, then the collected image was observed under the fluorescence microscope.

Total protein extracts were prepared using a lysis buffer (50 mM Tris, 2% SDS, 10% glycerol, 0.74 M beta-mercaptoethanol), sonicated on ice using a Sonifier 250D (Branson Ultrasonics, St. Louis MO, USA), and heated for 5 min at 99 °C. Protein concentrations were determined with the Bio-Rad DC Protein Assay Kit (BioRad, Hercules, CA, USA) using bovine serum albumin as a standard. Protein extracts were separated in 10% SDS-PAGE, electrotransferred to PVDF membranes (Immobilon-P; EMD Millipore, Burlington, MA, USA). Antibodies used are: HSP90 antibody [H90-10] #ab53497, MYC #ab32 (Abcam, Cambridge, UK) [36 (link)], #sc764 (Santa Cruz Biotechnology, Dallas, TX, USA) [37 (link)]; TUBULIN #ab6046 (Abcam, Cambridge, UK) [38 (link)]; Anti-MOUSE HRP #31430 (Thermo Fisher, Waltham, MA, USA) [39 (link)]; Anti-RABBIT HRP #31460 (Thermo Fisher, Waltham, MA, USA) [40 (link)].

Fetal bovine serum (FBS), Dulbecco’s modified Eagle’s medium (DMEM), and antibiotics were from HyClone (Logan, UT, USA). Transwell inserts with an 8 μm polycarbonate membrane and cell culture plastic labware were purchased from Corning Inc. (New York, NY, USA). Collagen IV was a product of Trevigen Inc. (Gaithersburg, MD, USA). Antibodies directed to Hsp90α (ab59459) and Hsp90β (ab53497) were purchased from Abcam (Cambridge, UK). Secondary Alexa 488-labeled conjugates were from Jackson ImmunoResearch Laboratories Inc. (West Grove, PA, USA). All other chemicals were from Sigma-Aldrich (St. Louis, MO, USA).