Rabbit anti hsp90

Goat anti-HSP27 (M-20), and mouse anti-αA-crystallin (B-2) antibodies were obtained from Santa Cruz Biotechnology (Dallas, TX, USA). Rabbit anti-vimentin, rabbit anti-histone H3, rabbit anti-MEK1/2, rabbit anti-HSP40, rabbit anti-HSP60, rabbit anti-HSP70, rabbit anti-HSP90, rabbit anti-AIF, goat anti-rabbit IgG-HRP-linked and horse anti-mouse IgG-HRP-linked antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA). Mouse anti-FLAG (M2) and mouse anti-β-actin antibodies were obtained from Millipore Sigma (St. Louis, MO, USA). Rabbit αB-crystallin antibody was obtained from Enzo Life Sciences (Farmingdale, NY, USA). Affinity purified anti-FAIM antibody was obtained from rabbits immunized with CYIKAVSSRKRKEGIIHTLI peptide (located near the C-terminal region of FAIM) as previously described [33 (link),46 (link)].

HeLa cells were lysed in RIPA buffer (150 mM NaCl, 50 mM Tris with pH 7.4, 1% Triton, 0.5% Nonidet P40, 10% glycerol, and 2.5% sodium deoxycholate) plus protease and phosphatase inhibitors (Roche Diagnostic, Germany, 11836153001). Protein concentrations were determined with the Bio-Rad Protein Assay Dye kit (Bio-Rad, United States, 5000006). Cell extracts were resolved by SDS-PAGE and transferred onto PVDF membranes (Millipore, Germany, Immobilon-P IPVH00010). Blocking was performed at room temperature in TBS 1X 0.1% Tween 5% low-fat milk for 1 h. Membranes were incubated with primary antibodies overnight at +4°C, followed by incubation with horseradish peroxidase-conjugate secondary antibodies (Bio-Rad) and revealed with western chemoluminescent HRP substrate (Millipore, Immobilon WBKLS0500). Chemoluminescent signals were acquired with the iBright CL1000 Imaging System (Thermo Fisher, United States). Quantitative analysis was performed using ImageJ software. Primary antibodies used were rabbit anti-p62 (MBL International Corporation, United States, PM045), mouse anti-COXII (Abcam, United Kingdom, ab110258), mouse anti-COXIV (Abcam, United Kingdom, ab33985), rabbit anti-LC3 A/B (Cell Signaling, United States, D3U4C, 12741S), rabbit anti-HSP90 (Cell Signalling; United States, E289, 4875), and mouse anti-vinculin (Santa Cruz Biotechnology, United States, 7F9, sc-73614).

For Western blot analysis, after extraction, 20 μg of total proteins was blotted onto nitrocellulose membranes and incubated with rabbit anti-Hsp90 (1:1,000; Cell Signaling Technologies, Danvers, MA, USA) and rabbit anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:20,000; Gentex, Zeeland, MI, USA), followed by incubation with horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (1:2,500; Santa Cruz Biotechnology, Dallas, TX, USA). Revelation and densitometric blots were performed in three different experiments.