Total cell lysates were prepared from biopsy protein following Qiazol (QIAGEN) RNA extraction. Briefly, protein pellets were resuspended in Laemmli buffer, incubated at 37°C for 30 minutes, vortexed briefly, and heated to 95°C for 5 minutes before electrophoresis. Cells were lysed using MPER lysis buffer (Sigma-Aldrich, St. Louis, MO) supplemented with protease inhibitors (Roche) and sodium orthovanadate (10 mM) (Sigma-Aldrich). Loading buffer (Life Technologies) was added and samples were heated to 95°C for 5 minutes, subjected to electrophoresis on 4-12% NuPAGE Bis-Tris gels (Life Technologies), transferred to nitrocellulose membranes (Life Technologies), and visualized using the Odyssey CLx system (LI-COR Biosciences, Lincoln, NE) with IRDye 800RD goat anti-rabbit (LI-COR) and IRDye 680RD goat anti-mouse (LI-COR Biosciences) secondary antibodies. Primary antibodies were: anti-LRRC31 (ab100379; Abcam PLC, Cambridge, UK) and anti-HSP90 (AF7247; R&D Systems, Minneapolis, MN). Blots were quantified using Image Studio (LI-COR).
Anti hsp90
Manufactured by R&D Systems
Sourced in United Kingdom, Germany
Anti-HSP90 is a laboratory reagent that detects the presence of the Heat Shock Protein 90 (HSP90) in biological samples. HSP90 is a molecular chaperone protein that plays a crucial role in the folding, stability, and function of various client proteins. The Anti-HSP90 product is designed to facilitate the identification and quantification of HSP90 in research and analytical applications.
Automatically generated - may contain errors
Lab products found in correlation
Nitrocellulose membrane, by Thermo Fisher Scientific (2 mentions)
Loading buffer, by Thermo Fisher Scientific (2 mentions)
Nupage bis tris gel, by Thermo Fisher Scientific (2 mentions)
Odyssey clx system, by LI COR (2 mentions)
Mper lysis buffer, by Merck Group (2 mentions)
Irdye 800rd goat anti rabbit, by LI COR (2 mentions)
Anti lrrc31 ab100379, by Abcam (2 mentions)
Image studio, by LI COR (2 mentions)
Irdye 680rd goat anti mouse, by LI COR (2 mentions)
Sodium orthovanadate, by Merck Group (2 mentions)
Qiazol, by Qiagen (2 mentions)
Anti erk1 2, by R&D Systems (2 mentions)
Anti rat 7077, by Cell Signaling Technology (2 mentions)
Anti phospho erk1 2, by Cell Signaling Technology (2 mentions)
Anti actin antibody, by MP Biomedicals (1 mentions)
Anti hsp60, by Merck Group (1 mentions)
Anti hsp70, by Stressgen (1 mentions)
Anti ha clone 12ca5, by Roche (1 mentions)
Sligkv nh2, by Bachem (1 mentions)
U0126, by Merck Group (1 mentions)
5 protocols using anti hsp90
1
Western Blot Analysis of Protein Lysates
2
Western Blot Analysis of Protein Lysates
3
Characterization of Prostate Cancer Cell Lines
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CA LNCaP, Du145, and PC3 cells were obtained from ATCC, while E006AA and E006AA-hT, AA prostate cancer cell lines were established and characterised by Dr Koochekpour's laboratory (Koochekpour et al, 2004 (link), 2014 (link)). Cells were maintained as recommended by ATCC. Primary antibodies against HSP10 and cell cycle and apoptotic cocktail (pCdk/pHH3/Actin/PARP), anti-MMP-9, anti-MMP-2 were from Abcam, MA, USA; anti-HSP60 was from Chemicon; anti-HSP90 from R&D Systems USA, anti-HSP70 from Stressgen, USA; and anti-actin antibody was from M.P. Biomedical USA. Rabbit and mouse HRP-labelled secondary antibodies were procured from GE Healthcare Life Sciences, Pittsburgh, PA, USA. All other reagents were purchased from Sigma-Aldrich, St. Louis, MO, USA.
4
Pharmacological Inhibitors for Cell Signaling
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The MEK inhibitor U0126 and the Rac1 inhibitor NSC23766 were purchased from Calbiochem/Merck. GB88 and GB110 were a kind gift of Dr. David B. Fairlie (The University of Queensland, Brisbane, Australia) [33 (link),34 (link)]. The PAR2-selective peptide agonist SLIGKV-NH2 and the PAR1-selective agonist peptide TFLLRN-NH2 (STRAP-1) were obtained from Bachem (Bubendorf, Switzerland). Synthesis, coupling, cleavage from the resin and characterization of the PAR2-selective peptide 2-furoyl-LIGRLO-NH2 (2f-LI, EC50 = 2.5 μM) were done as described in detail before [26 (link)]. The following primary antibodies were used: anti-ALK5 antibody (TGFβ RI (V22), Santa Cruz Biotechnology, Santa Cruz, CA), anti-phospho-ERK1/2 (#4370, Cell Signalling Technology, Frankfurt/Main, Germany), anti-HSP90 (both #sc-7947 and #sc-13119), anti-ERK1/2 (#AF1576, R&D Systems, Wiesbaden, Germany), and anti-HA (clone 12CA5, Roche, Mannheim, Germany). HRP-linked anti-rabbit (#7074), anti-mouse (#7076), and anti-rat (#7077) secondary antibodies were from Cell Signaling Technology. The rhTGF-β1 (#300-023) was purchased from ReliaTech (Wolfenbüttel, Germany) and used at a concentration of 5 ng/mL.
5
Western Blot Analysis of Signaling Pathways
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The following primary antibodies were used: Anti-phospho-ERK1/2 (#4370, Cell Signalling Technology, Frankfurt/Main, Germany), anti-HSP90 (both #sc-7947 and #sc-13119), anti-ERK1/2 (#AF1576) and phospho-Smad3(Ser423/425) (#AB3226), both from R&D Systems, Wiesbaden, Germany anti-Rac1b (#09-271, Merck Millipore, Darmstadt, Germany), anti-Rac1 (#610650), anti-CIP1/WAF1 (#610233) (both from BD Biosciences, Heidelberg, Germany), anti-proteasome (#PW-8195, Biomol, Plymouth Meeting, PA, USA), anti-HSP27 (#SA-348, Biomol), anti-MnSOD (#06-984, Upstate Cell Signaling Solutions, Lake Placid, NY, USA), anti-osgin (#H00029948-B01P, Abnova, Taipei, Taiwan), anti-β-actin (#A1978, Sigma-Aldrich, Deisenhofen, Germany). HRP-linked anti-rabbit (#7074), anti-mouse (#7076) and anti-rat (#7077) secondary antibodies were from Cell Signaling Technology, anti-goat secondary antibody (#ab6741) was from Abcam (Cambridge, UK). The rhTGF-β1 (#300-023) was purchased from ReliaTech (Wolfenbüttel, Germany) and used at a concentration of 5 ng/mL for the breast cancer cell lines and 10 ng/mL for HMEC.
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