Goat anti mouse igg horseradish peroxidase

Western blot analysis was performed as described previously (Ko et al., 2012). Blotted membranes were immunostained with antibodies specific for the following antigens: HA tag (Covance, New York, NY, USA); Myc tag, Smad2/3, phosphoSmad2/3 and carboxyl terminus of Hsc70‐interacting protein (CHIP; Cell Signaling, Danvers, MA, USA); Flag tag and β‐actin (Sigma‐Aldrich); 6 × His tag and CK2β (R&D Systems); E‐cadherin and N‐cadherin (Thermo Fisher Scientific, Rockford, IL, USA); WW domain containing E3 ubiquitin protein ligase 1 (WWP1) (ProteinTech Group, Inc., Chicago, IL, USA); CK2α (EMD Millipore, Burlington, MA, USA); and HDAC I, Smad4 and vimentin (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA). The secondary antibodies were goat anti‐rabbit IgG peroxidase, goat anti‐mouse IgG horseradish peroxidase (Thermo Fisher Scientific) and donkey anti‐sheep IgG horseradish peroxidase (R&D Systems). Signals were developed using Lumi‐Light Western Blotting Substrate (Roche Diagnostics, Indianapolis, IN, USA) in accordance with the manufacturer's instructions.

Protein lysate preparation and western blot analysis were done as described by Kenche et al.^{36 (link)}. The following primary antibodies were used: α-Ago2 (Wako Chemicals, Cat. # 018-22021, 1:500), α-tubulin (Millipore-Sigma, Cat. # T9026, 1:5000), α-lactate dehydrogenase A, (Cell Signaling, Cat # 2012, 1:1000), α-succinate dehydrogenase subunit A, (Abcam, Cat # ab14715, 1:1000), α-H3 (Cell Signaling, Cat # 9715, 1:4000), α-GADD34 (Santa Cruz, Cat. # SC-825, 1:500), α-Actin (Abcam, Cat. # ab6276, 1:10,000), α-Phospho-eIF2α (Ser51) (Cell Signaling, Cat. # 3597, 1:1000), α-eIF2α total (Cell Signaling, Cat. # 9722, 1:1000), α-Trib3 (LSBio, LifeSpan BioSciences, Cat. # LS-C164592, 1:250), α-Phospho-Akt (Trh308), (Cell Signaling, Cat. # 2965, 1:500), and α-Akt total (Cell Signaling, Cat. # 9272, 1:1000). Secondary antibodies were goat anti-mouse IgG Horse Radish Peroxidase (Thermo Fisher Scientific, Cat # 31430, 1:5000), and goat anti-rabbit IgG Horse Radish Peroxidase (Thermo Fisher Scientific, Cat # 31460, 1:5000). All original blots are provided in Supplementary Fig. 5.

The inhibitors for specific Hsp90 isoforms [KUNA115 and NDNA1065 (Hsp90α), KUNB106 and its analog, NDNB1151 (Hsp90β), KUNG65 (Grp94), T1, T2, and T3 (TRAP1)] were synthesized and purified by the laboratory of Dr. Brian Blagg at the University of Notre Dame [(Crowley et al., 2017 (link); Mishra et al., 2021a (link); Mishra et al., 2021b (link); Merfeld et al., 2023 (link)), Figure 1]. Lipopolysaccharide (LPS, from E. coli O111:B4), 17-AAG and penicillin:streptomycin (tissue culture) were purchased from VWR (Radnor, PA, United States). Dulbecco’s Modified Eagle Medium (DMEM), trypsin-EDTA, fetal bovine serum (FBS), and G418 disulfate solution were obtained from Corning (Corning, NY, United States). Griess reagents for nitric oxide assay (sodium nitrite, sulfanilamide, N-1-naphthylethylenediamine) were purchased from Sigma-Aldrich (St. Louis, MO, United States). Antibodies used for Western blots include goat anti-rabbit IgG-horseradish peroxidase, and goat anti-mouse IgG-horseradish peroxidase (Invitrogen, Waltham, MA, United States); p-ERK, t-ERK, p-p38, t-p38, p-JNK, and t-JNK (Cell Signaling, Beverly, MA, United States).