Hsp90α β f 8

Manufactured by Santa Cruz Biotechnology

HSP90α/β (F-8) is a mouse monoclonal antibody that recognizes the alpha and beta isoforms of the heat shock protein 90 (HSP90) family. HSP90 is a highly conserved molecular chaperone involved in the folding, stabilization, and activation of various client proteins.

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4 protocols using hsp90α β f 8

Western Blot Analysis of Circadian and Inflammatory Proteins

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Protein of cultured cells and liver tissue was extracted using RIPA buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 1% Triton X-100, 0.5% sodium deoxycholate containing protease and phosphatase inhibitors). Protein samples (40 μg) were subjected to electrophoresis on 10% SDS-polyacrylamide gels and transferred to a nitrocellulose (Amersham Protran Supported 0.45 NC, GE healthcare). After blocking with 0.5% dry milk in PBS-Tween 0.1%, the membranes were probed with antibodies against GR (1:500, Santa Cruz Biotechnology, sc-1004), HSP90α/β (F-8) (1:1000, Santa Cruz Biotechnology, sc-13119), REV-ERBα (1:200, Santa Cruz Biotechnology, sc-100910), BMAL1 (1:500, Ripperger and Schibler, 2006 (link)), NF-κB p65 (1:200, Santa Cruz Biotechnology, sc-109), IκBα (1:200, Santa Cruz Biotechnology, sc-371), phosphorylated (p-)IκBα (1:200, Santa Cruz Biotechnology, sc-8404), tubulin (1:1000, Abcam, ab 15246) or LAMINB1 (1:500, Santa Cruz Biotechnology, sc-30264) overnight at 4°C. Anti-rabbit-IgG, mouse-IgG and goat-IgG antibody conjugated to horseradish peroxidase (HPR) was used as a secondary antibody. Detection of the immune complexes was performed using Western Bright Quantum system (Advansta) and quantification was done with the Quantity One analysis software (BioRad).

Okabe T., Chavan R., Fonseca Costa S.S., Brenna A., Ripperger J.A, & Albrecht U. (2016). REV-ERBα influences the stability and nuclear localization of the glucocorticoid receptor. Journal of Cell Science, 129(21), 4143-4154.

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Base Editing Protein Expression Analysis

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To analyze protein expression during a base editing experiments, the transfection and base editing protocol was performed as described above using intact BE4max or seBE constructs and the EMX1-targeting sgRNA plasmid. At the end of the experiment, cells were resuspended in CytoBuster Protein Extraction Reagent (Millipore Sigma) for lysis according to manufacturer’s instructions. Protein concentration was quantified by Qubit Protein Assay Kit (ThermoFisher), and 40 µg of total protein was loaded into a 4–15% Mini-Protean TGX Precast Protein Gel (BioRad). After electrophoresis, the iBlot Dry Blotting System (ThermoFisher) was used for transfer onto PVDF. The membrane was then blocked with 5% (w/v) low fat milk, 20 mM Tris-HCl, 10 mM NaCl and 0.1% Tween-20 (TBST) and incubated at 4°C with the appropriate primary antibody overnight: Myc-Tag (9B11) Mouse mAb (Cell Signaling) at 1:2000 dilution, anti-Cas (7A9–3A3) (Cell Signaling) at 1:1000 or Hsp90α/β (F-8) at 1:200 (Santa Cruz Biotechnology). The next day, the membranes were washed in 1X TBST and incubated in blocking buffer at 4°C for 1 hour with m-IgGκ BP-HRP secondary antibody (Santa Cruz Biotechnology). The membranes were imaged using Immobilon Western Chemiluminescent HRP Substrate (Millipore Sigma).

Berríos K.N., Evitt N.H., DeWeerd R.A., Ren D., Luo M., Barka A., Wang T., Bartman C.R., Lan Y., Green A.M., Shi J, & Kohli R.M. (2021). CONTROLLABLE GENOME EDITING WITH SPLIT-ENGINEERED BASE EDITORS. Nature chemical biology, 17(12), 1262-1270.

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Exosomal Protein Profiling by Western Blot

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Alix antibody (cat. no. ab117600, Abcam), α-actinin antibody (H-2, cat. no. sc-17829, Santa-Cruz), β-actin antibody (Clone AC-74, cat. no. A2228, Sigma-Aldrich), Calnexin antibody (Clone C5C9, cat. no. 2679, Cell Signaling), CD63 antibody (cat. no. 25682–1-AP, Proteintech), HSP90α/β (F-8, cat. no. sc-13119, Santa-Cruz), TSG101 antibody (Clone 4A10, cat. no. MA1-23296, Thermo Fisher).
Western blots were used to examine the presence of common exosomal proteins in cellular fractions and sucrose gradient purified exosomes. Using Capan-2 cells as a representative example, equivalent micrograms of proteins from ER and mitochondrial (P2), cytoplasmic (S2), media (M), and exosome (Ex) fractions, prepared from different steps during exosome isolation as described above in section 1.1, were separated by SDS-PAGE and transferred to nitrocellulose membranes. Development was performed using Pierce ECL 2 Western blotting substrate (Thermo Fisher Scientific) and radiographic films (Lightlab).

Stefanius K., Servage K., de Souza Santos M., Gray H.F., Toombs J.E., Chimalapati S., Kim M.S., Malladi V.S., Brekken R, & Orth K. (2019). Human pancreatic cancer cell exosomes, but not human normal cell exosomes, act as an initiator in cell transformation. eLife, 8, e40226.

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Western Blot Analysis of SHIP1 Protein

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Cells were harvested from an 80% confluent P2 culture. After two washes with cold PBS, cells were spun down at 10,000xg for 1 min and lysed on ice for 20 min with Lysis buffer (20mM Tris, 150mM NaCl, 1mM EGDA, 1mM EGTA, 1%Triton X-100) containing 1x protease and phosphatases inhibitor Halt (ThermoFisher Scientific). The cell lysate was spun down for 20 min at 10,000xg to eliminate cell debris. Western blot was then performed as described previously (Pedicone et al., 2019 (link)). Briefly, 25μg of cleared lysate in loading buffer containing DTT was loaded on 4-15% Precast gels (Bio-Rad). The gel was run 30 min at 180V followed by transfer to a nitrocellulose membrane with Trans-Blot Turbo (Bio-Rad). The membrane was blocked in 5% Nonfat milk (Cell Signaling) in TBST (Tris-Buffer Saline, 0.1% Tween 20) O/N and incubated 2h with 1:500 SHIP1 P1C1 (Santa Cruz Biotechnology), 1:1000 β-actin C-4 (Santa Cruz Biotechnology), or 1:1000 HSP90 α−β F-8 (Santa Cruz Biotechnology). After three-10-min washes in TBST the membrane was incubated with1:1000 m-IgGkBP-HRP (Santa Cruz Biotechnology) in 5% Nonfat milk for 45 min. To detect the chemiluminescence signal, we used a Bio-Rad Chemidoc with ECL Pico(ThermoFisher Scientific) for housekeeping proteins and ECL Femto (ThermoFisher Scientific) for SHIP1.

Pedicone C., Fernandes S., Matera A., Meyer S.T., Loh S., Ha J.H., Bernard D., Chisholm J.D., Paolicelli R.C, & Kerr W.G. (2022). Discovery of a novel SHIP1 agonist that promotes degradation of lipid-laden phagocytic cargo by microglia. iScience, 25(4), 104170.

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