Anti hsp90 antibody

Cell lysates were prepared with cell lysis buffer (20 mM Tris-Cl pH7.4, 25 mM NaCl and 0.1% NP40) containing proteinase inhibitors. Proteins (2 mg) were incubated with control IgG, monoclonal anti-STIP1antibody (Abnova) or anti-HSP90 antibody (Abcam) at 4°C overnight under agitation. Enriched protein complexes were concentrated with protein A agarose beads (Millipore, Billerica, MA, USA). Agarose beads were washed for three times with wash buffer (20mM Tris-Cl pH7.4, 25mM NaCl) and boiled with sample buffer (0.25 M Tris-Cl pH 6.8, 0.08% SDS, 20% glycerol, and 10% β-mercaptoethanol). Immunoprecipitation was performed with an ImmunoCruz IP/WB optima system (Santa Cruz Biotechnology). Each sample was subjected to electrophoresis with 8% SDS-PAGE. Antibodies raised against JAK2, HSP90 (Cell Signaling Technology), STAT3 (Abcam), and STIP1 (Abnova) were used for western blot analysis. Halo fusion proteins, NTAP proteins, and His-tagged ubiquitin were pulled down using the Halo-tag protein purification system sample pack (Promega), streptavidin agarose (Sigma) and nickel beads (Invitrogen), respectively, following the manufacturer's protocols.

Cytokines were measured in EVs isolated from 5 × 10⁵ human monocytes, 5 × 10⁵ human M1 macrophages, 1 × 10⁶ mouse monocytes, or 1 × 10⁷ human/mouse whole-blood cells. TGF-β1 was measured with Human TGF-β1 DuoSet ELISA (R&D Systems, cat no. DY240) and mouse TGF-β1 DuoSet ELISA (R&D Systems, cat no. DY1679) or Human/Mouse TGF-β1 ELISA Ready-SET-Go! Kit (eBioscience, cat no. 88-8350-88). IL-10, IL-6, and Il-1β were measured using the human IL-10 ELISA Ready-SET-Go! Kit (eBioscience, cat no. 88-7106-22), human IL-6 ELISA Ready-SET-Go! Kit (eBioscience, cat no. 88-7066-22), or human IL-1β ELISA Ready-SET-Go! Kit (eBioscience, cat no. 88-7261-86). In all cases, the ELISA was performed according to the manufacturer’s protocol. CD9 and HSP90 were measured with ELISA on immobilized vesicle using anti-CD9 antibody (Novus Biologicals, cat no. NB500-327) (2 µg/mL) and anti-HSP90 antibody (Abcam, cat no ab79848) (2 µg/mL), respectively. Vesicles in different fractions of size-exclusion chromatography was immobilized with TGF-β1 capture antibody from Human TGF-β1 DuoSet ELISA, and CD9 or HSP90 was detected on vesicles by biotinylated CD9 and HSP90 antibody and streptavidin-conjugated HRP.

At the end of treatment, the culture media were aspirated and the cells were washed once with ice-cold PBS. The cells were then lysed with lysis buffer containing 20 mM Tris (pH 7.5), 150 mM NaCl, 1% Triton X-100, and sodium pyrophosphate, β-glycerophosphate, EDTA, Na₃VO₄, leupeptin, and other protease inhibitors (Sigma-Aldrich). Protein samples per 100 μg were added with 4 μL anti-HSP90 antibody (Abcam, Cambridge, UK) and shaked at 4°C overnight. 40 μL Protein A Agarose (Sigma Chemical Company) was added and incubated for 3 h at 4°C and then centrifuged at 1000 g for 5 min. After that, the supernatant was discarded and the pellet was washed 3 times with PBS. Thereafter, the precipitates were resuspended with 40 μL 1x sample buffer and heated at 96°C for 5 min. The samples were centrifuged for 5 s and the supernatants were collected for Western blotting.

CT26 colorectal cancer cells were treated with 10 μM IR-780 for 24 h, then CRT and HSP90 expression levels were determined using an immunofluorescence assay. After washing with PBS, cells were fixed in 4% of paraformaldehyde for 5 min and incubated with anti-calreticulin antibody (Cat# ab2907, Abcam) or anti-Hsp90 antibody (Cat# ab13492, Abcam) at 4°C overnight. After washing with PBS three times, cells were stained with secondary antibody (Cat# ab150077, Abcam) at 37°C for 40 min. Nuclei were stained with DAPI, then observed using fluorescence microscopy. CRT-positive cells were also analyzed by flow cytometry following staining. ATP and HMGB1 released in culture supernatants were detected using an enhanced ATP assay kit (Cat# S0027, Beyotime Biotechnology) and an HMGB1 ELISA kit (Cat# 6010, Chondrex), respectively, according to the manufacturer's instructions.

Total lysate (0.4 mg) from control and drug-treated cells was incubated overnight with 5 μl of anti-HSP90 antibody (Abcam, 13495) at 4 °C. Protein A/G magnetic beads (20 μl) were added to the mixture with incubation (gentle rotation) for 4 h at 4 °C. Immunoprecipitated samples were washed and subjected to Western blot analysis with anti-pan-acetyl-lysine antibody (Cell Signaling, 94415) and anti-HSP90 antibody.