Gapdh clone 6c5

Primary antibodies were: mouse monoclonal antibodies against Myc clone 9E10 (Sigma), Flotillin-2 (BD Biosciences), α-Synuclein (Invitrogen), Alix (BD Biosciences), TSG101 (GeneTex Inc., Irvine, CA, USA), CD63 (BD Biosciences), 6E10 anti-APP (Signet), GAPDH clone 6C5 (Abcam), rabbit anti-Glutamate Receptor GluR2/3 (Chemicon), rabbit anti-Glutamate Receptor GluR1 (Chemicon), rabbit anti-Calnexin (StressGene), rabbit anti-GFP (Invitrogen), rabbit anti-Integrin β5 (Millipore), rabbit anti-UBC9 (Santa Cruz), and rat anti-LAMP1 (1D4B) (Santa Cruz). SUMO-2 antibody was kindly provided by F. Melchior (Heidelberg, ZMBH, Germany) [5 (link)]. Secondary antibodies were obtained from Dianova and Invitrogen.

MSCWJ-NEU cells were incubated with IA-50 at a concentration of 1 µM for 24 h. Cells were then lysed by three freeze–thaw cycles in a low RIPA buffer containing 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.05% Tween-20, 0.1% SDS, 3 mM EDTA and 1 mM PMSF. To obtain lysates of hippocampal tissues, the mice were sacrificed, and the hippocampus was immediately removed and lysed in a low RIPA buffer. Lysates of MSCWJ-NEU cells or hippocampal tissues were subjected to electrophoresis, following Western. The membrane was subsequently incubated with antibodies against Hsp70, clone 3B5 [47 (link)] and glyceraldehyde-3-phosphate dehydrogenase, taken as a loading control (GAPDH, Clone 6C5, Abcam, Cambridge, UK).

Protein extraction was carried out as previously described.^{14 (link)} Fractionation of nuclear and cytoplasmic protein was carried out using the NE-PER^TM nuclear and cytoplasmic fraction reagent (Thermo Fisher Scientific) according to the manufacturer’s instructions. Fifteen micrograms of nuclear protein lysates and equivalent amount of the cytoplasmic protein lysates were separated on 4–15% Precast PAGE gels (Bio-Rad) and electrotransferred to nitrocellulose membranes. The membranes were probed with antibodies against PTPN14 (Sigma-Aldrich, NSW, Australia), YAP (clone D8H1X, Cell Signaling, MA, USA), Taz (clone V386, Cell Signaling), stathmin (BD Bioscience, Victoria, Australia), Topoisomerase I (Novus Biologicals, CO, USA) as a control for nuclear fraction and GAPDH (clone 6C5, Abcam, Victoria, Australia) as a control for equal loading. Proteins were detected by ECL Plus (Pierce, Thermo Fisher Scientific) and membranes were either scanned using the Typhoon (GE Healthcare) or exposed to the film. Densitometry analysis was carried out as previously described.^{15 (link)}

RIPA extracts (20–40 µg protein per lane) and purified His-tagged proteins from conditioned media (equivalent of 20 µl conditioned medium) were subjected to PAGE on 8–10 % polyacrylamide gels and western blotting according to standard procedures. Nitrocellulose blots (Whatman Optitran BA-S85, GE Healthcare, Little Chalfont, UK) were blocked with PBS/Odyssey Blocking Buffer (LI-COR Biosciences, Lincoln, NE, USA) (1:1), followed by overnight incubation with primary antibodies at 4 °C. Antibodies used were against MET N-terminus (clone EP1454Y, Epitomics, Abcam, Cambridge, UK), MET C-terminus (clone D1C2), phosphorylated (P)-MET (Y1234/1235, clone D26), P-AKT (S473, clone D9E), P-ERK1/2 (T202/Y204, clone 20G11) (all CST), GAPDH (clone 6C5, Abcam), and α-tubulin (clone 236-10501, Molecular Probes, Life Technologies). Biotin groups and primary antibodies were visualized using, respectively, streptavidin-680 (Molecular Probes, Life Technology) and appropriate secondary antibodies [goat-anti-rabbit-IRDye800 (Rockland Immunochemicals, Gilbertsville, PA, USA) or Alexa Fluor 680 goat-anti-mouse IgG (Molecular Probes, Life Technologies)]. Blots were scanned on the Odyssey imager (LI-COR Biosciences).

The reagents and their sources were as follows: bovine type II collagen, complete Freund’s adjuvant (CFA), and incomplete Freund’s adjuvant (IFA) were purchased from Chondrex (Redmond, WA). Recombinant human MMP-1, MMP-2, and MMP-3 were obtained from Chemicon (Temecula, CA), and recombinant human cathepsin D, cathepsin L, and cathepsin K were obtained from Calbiochem (La Jolla, CA). Polyclonal antibody was generated by immunizing rabbits with recombinant βig-h3. Antibodies against murine CD3 (Dako, Glostrup, Denmark), CD31 (clone MEC13.3; BD Bioscience, San Jose, CA), ICAM-1 (clone 166623) and receptor activator of nuclear factor kappa-B ligand (RANKL; clone 88227; R&D Systems, Minneapolis, MN), GAPDH (clone 6C5; Abcam, Cambridge, UK), human MMP-2 (Millipore, Billerica, MA), and the histidine-tag (Penta·His, Qiagen, Hilden, Germany) were used as primary antibodies. Secondary antibodies included horseradish peroxidase (HRP)-conjugated goat anti-rat and rabbit anti-goat, and biotinylated rabbit anti-rat and swine anti-rabbit antibodies (Dako).

C6 rat glioblastoma cells were incubated with PQ-29 at a concentration of 0.2, 0.5, and 1 μM for 18 h, lysed, and lysates were used for electrophoresis and blotting as described previously [28 (link)]. The blot was subsequently incubated with antibodies against Hsp70, clone 3C5 [29 (link)], and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, Clone 6C5, Abcam, Cambridge, UK).
To analyze Hsf1 trimers, we applied non-denaturating gradient polyacrylamide gel electrophoresis. The gradient gel was prepared by layering partially blended 4% and 15% solutions from 30% stock of 37:1 acrylamide:bis-acrylamide mixture. Early obtained cell lysates with a concentration of 80 mg were loaded into the gradient gel without adding β-mercaptoethanol, SDS and without heating. Electrophoresis buffer excluded SDS.