Goat anti rabbit igg h l pe preadsorbed

Cells were stained with anti-human TRAP1-RPE (3H4-2H6, Sigma-Aldrich), primary antibody against HSP90β (EPR16621), STIP1 (EPR6605) and the secondary antibody goat anti-rabbit IgG H&L PE preadsorbed (all Abcam). Mouse IgG1-PE (Invitrogen) and PE-rabbit IgG (Abcam) were used as isotype controls. FcR blocking reagent (Miltenyi Biotec) was used to block non-specific binding. For intracellular staining, cells were fixed and permeabilized with Cytofix/Cytoperm (BD Biosciences) and stained with antibodies for intracellular proteins. For surface and intracellular staining, dead cells were excluded from gating with the use of Sytox Blue dead stain and Fixable Viability Dye eFluor 506 (Invitrogen), respectively.

T cells were stained with primary antibodies against HSP90α (2G5.G3, Abcam, USA), HSP90β (EPR16621, Abcam, USA), HSP70 (C92F3A-5, Enzo Life Sciences, USA), HSC70 (1B5, Stressgen, USA), HSP40/DNAJB1 (3B9.E6, Abcam, USA), GRP78 (StressMarq Biosciences, USA), and HSP60 (StressMarq Biosciences, USA). Secondary antibodies were goat anti-rabbit IgG H&L (PE) preadsorbed (Abcam), anti-mouse IgG1 (PE) (Miltenyi Biotec, Germany), and anti-rat F(ab’)2 (PE) (Sony Biotechnology, USA). T cells were also stained with anti-human CD279/PD-1 (REA1165, Miltenyi Biotec, Germany) (PE), CD152/CTLA-4 (REA1003, Miltenyi Biotec, Germany) (PE), primary antibody against STAT3 (4G4B45, Sony Biotechnology, USA), and secondary antibody anti-mouse IgG1 (PE) (Miltenyi Biotec, Germany). The FcR blocking reagent (Miltenyi Biotec, Germany) and isotype controls were used to exclude non-specific binding. For intracellular staining, cells were fixed and permeabilized using Cytofix/Cytoperm. Cells were analyzed by flow cytometry.