Western blot using BmN4 cell lysates was performed as described previously36 (link). Cell lysates were prepared from identical number of the cells from respective density conditions in a lysis buffer containing 20 mM Tris-HCl pH 7.4, 200 mM NaCl, 2.5 mM MgCl2, 0.5% NP-40, 0.1% Triton X-100, and complete protease inhibitor (Roche Diagnostics). After quantifying protein concentration of the lysates, 20 μg of each lysate was loaded onto SDS-PAGE. The following antibodies were used: S213 anti-Siwi36 (link), anti-BmAgo323 (link), anti-Tom20 (Santa Cruz Biotechnology), anti-Hsp60 (Cell Signaling), anti-β-actin (Abcam), anti-BmPapi10 (link), and BmVasa571 anti-BmVasa53 . The anti-BmVasa is able to specifically recognize BmVasa in Western blot and immunofluorescence (Supplementary Fig. S1 ). The Western blot band intensities showed clear linearity to the amount of lysate input (2.5–20 μg), suggesting the quantification ability of the Western blot analyses (Supplementary Fig. S2 ).
Anti hsp60
Manufactured by Cell Signaling Technology
Sourced in United States, United Kingdom
Anti-HSP60 is a laboratory reagent that detects the heat shock protein 60 (HSP60) in cellular samples. HSP60 is a molecular chaperone involved in protein folding. Anti-HSP60 can be used to identify and quantify HSP60 expression levels in various experimental systems.
Automatically generated - may contain errors
Lab products found in correlation
Anti actin, by Merck Group (3 mentions)
Anti tom20, by Santa Cruz Biotechnology (2 mentions)
Anti β actin, by Abcam (2 mentions)
Anti flag, by Merck Group (2 mentions)
Anti hsp40, by Cell Signaling Technology (2 mentions)
Horseradish peroxidase conjugated secondary antibody, by Thermo Fisher Scientific (2 mentions)
Protease inhibitor, by Roche (2 mentions)
Ai600 imager, by Cytiva (2 mentions)
Anti opa1, by BD (2 mentions)
Anti β tubulin, by Developmental Studies Hybridoma Bank (1 mentions)
Anti mcl 1, by Santa Cruz Biotechnology (1 mentions)
Anti p mek1 2, by Cell Signaling Technology (1 mentions)
Anti bcl 2, by Santa Cruz Biotechnology (1 mentions)
Anti bak, by Santa Cruz Biotechnology (1 mentions)
Hrp substrate, by Thermo Fisher Scientific (1 mentions)
Hrp conjugated secondary antibody, by Cell Signaling Technology (1 mentions)
Anti erk1 2, by Cell Signaling Technology (1 mentions)
Anti bax, by Santa Cruz Biotechnology (1 mentions)
Anti p erk1 2, by Cell Signaling Technology (1 mentions)
Anti β actin, by Cell Signaling Technology (1 mentions)
16 protocols using anti hsp60
1
Western Blot Analysis of BmN4 Cell Lysates
2
Western Blot Analysis of Testis Proteins
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The BmN4 cell lysates were subjected to western blots as described previously15 (link),34 (link). The following antibodies were used: 17.8 anti-Mili (1:1000)56 (link), BMK5263 anti-BmRNase κ (1:500), S213 anti-Siwi (1:1000)15 (link), anti-BmAgo3 (1:500)29 (link), anti-BmPapi (1:1000)11 (link), BmVasa571 anti-BmVasa (1:500)34 (link),60 , anti-Hsp60 (Cell Signaling Technology, #4870, 1:2000), anti-Tom20 (Santa Cruz Biotechnology, SC-11415, 1:1000), anti-β-Actin (Abcam, ab8224, 1:2000), anti-β-Tubulin (Developmental Studies Hybridoma Bank, E7, 1:2000), and anti-FLAG (Sigma, F3165, 1:1000).
3
Evaluating Cellular Stress Markers in Breast Cancer Cell Lines
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MDA-MB-468 cells and MCF-7 were treated with the indicated concentrations of ChPL for 24 h, and Western blot analysis was performed according to the previously published procedure (Kawiak et al., 2012b (link)). The preparation of cytosolic and mitochondrial fractions for cytochrome c release evaluation was performed as previously described (Kawiak et al., 2012b (link)). The following specific primary antibodies were used: anti-β-actin (1:1,000) (Cell Signaling, Danvers, MA, USA), anti-Bcl-2, anti-Bak, anti-Bax, and anti-Mcl-1 (1:250) (Santa Cruz, Heidelberg, Germany), anti-ERK1/2, anti-MEK1/2, anti-p-ERK1/2, and anti-p-MEK1/2 (1:1,000) (Cell Signaling), anti-cytochrome c (1:5,000) (Abcam, UK), and anti-HSP60 (1:1,000) (Cell Signaling). Membranes were incubated with primary antibodies overnight at 4°C after which a 1-h incubation with HRP-conjugated secondary antibodies (1:2000) (Cell Signaling) was carried out. Protein levels were determined by chemiluminescence (ChemiDoc; Bio-Rad, Waltham, MA, USA) with a HRP substrate (Thermo Scientific, MA, USA).
4
Immunofluorescence Staining and Confocal Microscopy
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Immunofluorescence staining was performed with anti-CD68 (25747-1-AP, Proteintech, 1:200) using a PANO 4-plex IHC kit (Yuanxi, Shanghai, China) as previously described 19 (link). MEFs were stained with anti-p-STAT3 (Tyr705) (D3A7, cat# 9145, 1:200), anti-p-STAT3 (Tyr727) (cat# 9134, 1:200), anti-HSP60 (D6F1, cat# 12165, 1:1400) using the APExBIO kit (APExBIO, Houston, USA). Anti-p-STAT3 (Tyr705), anti-p-STAT3 (Tyr727) and anti-HSP60 antibodies were purchased from Cell Signaling Technology (MA, USA). The analyses were carried out under a Zeiss LSM880/800 confocal microscope.
5
Immunoblotting Analysis of Cellular Stress Responses
6
Generation and Characterization of Parkin Antibodies
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Rabbit anti-Drosophila Parkin polyclonal antibody was raised against recombinant MBP-tagged Drosophila Parkin (275–482 aa) produced in the E. coli strain Rosetta 2 (Novagen). The antibodies used in the western blot analysis were as follows: anti-Parkin (1∶5,000 dilution), anti-Mfn (1∶2,000 dilution; a kind gift of Dr A. Whitworth), anti-Miro (1∶2,000 dilution; a kind gift of Dr E. Zinsmaier), anti-Drp1 (1∶2,000 dilution; a kind gift of Dr L. Pallanck), anti-ATP5A (1∶10,000 dilution; Abcam, 15H4C4), anti-NDUFS3 (1∶10,000 dilution; Abcam, 17D95), anti-Actin (1∶10,000 dilution; Millipore, MAb1501) and anti-Hsp60 (1∶1,000 dilution; Cell Signaling, D307). The antibody used in immunocytochemistry was anti-TH (1∶1,000 dilution; ref. [11] (link)). Complementary DNAs for human and Drosophila Parkin and PINK1 were described in previous studies [11] (link), [14] (link), [45] (link). Parkin phospho-mutants were generated by PCR-based mutagenesis followed by sequence confirmation of the entire gene.
7
Mitochondrial Morphology Quantification in Infected Cells
8
Western Blot Analysis of Cellular Proteins
9
Mitochondrial Dynamics Protein Quantification
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Cells at the same confluency were collected and RIPA buffer (Thermo Scientific™, 89900) complimented with protease inhibitors was used to lyse and collect the protein extracts. Following quantification of protein concentrations, 50 μg of total protein from control and patient fibroblasts were loaded on an SDS-PAGE gel. Subsequently, PVDF membranes were used for overnight transfer of the blots. The blots were probed with the following antibodies: anti-MFN2 (Abnova, H00009927-M03; 1:1000), anti-MFN1 (Cell Signaling Technology, D6E2S; 1:1000), anti-OPA1 (BD Transduction Laboratories, 612607; 1:1000), anti-Actin (Sigma, A5316; 1:1000), anti- VDAC1 (abcam, ab14734; 1:1000), anti-HSP60 (Cell Signaling Technology, D6F1; 1:1000). Complimentary horseradish peroxidase conjugated antibodies were used: goat anti rabbit IgG, HRP linked Antibody (Cell Signaling Technology, 7074S) or goat anti-mouse IgG-HRP (Santa Cruz Biotechnology, sc-2055). Blots were treated with the SuperSignal™ West Femto Maximum Sensitivity Substrate (Thermo Scientific™, 34095), and luminescence visualized with an Amersham Imager AI600.
10
Mitochondrial Protein Extraction and Analysis
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Purified mitochondria were incubated with protease K (for 25 min at 0°C) to remove the cytosolic proteins loosely associated with the outer mitochondrial membrane. The purified mitochondrial pellet was lysed in a buffer containing 50 mmol/L HEPES, 100 mmol/L NaCl, 1% NP-40, and a mixture of protease inhibitors (Roche Molecular Biochemicals, Mannheim Germany) and phosphatase inhibitors (Sigma). After homogenizing with 20 strokes using a Dounce homogenizer ( Bellco Glass, Vineland, NJ, U.S.A.), mitochondrial lysates were centrifuged at 20,000 g, and the supernatants were used for western blot analysis with anti-PGC-1α,25 (link) anti-SIRT1 (sc-15404; Santa Cruz Biotechnology, Dallas, Texas, U.S.A.), anti-TFAM (NBP1-71648; Novus Biologicals, Littleton, CO, U.S.A.), anti-pTau Ser404 (sc-12952; Santa Cruz Biotechnology), anti-pTau Ser396 (44752; Invitrogen), anti-pTau Ser262 (ab131354; AbCam, Cambridge, MA. U.S.A.), anti-pTau Thr 231 (AB 9668; Millipore, Billerica, MA, U.S.A.), anti-Histone H3 (ab1791; AbCam), anti-GAPDH (2118; Cell Signaling), or anti-HSP 60 (4870; Cell Signaling) antibodies. Horseradish peroxidase-conjugated secondary antibodies were purchased from Pierce Biotechnology, Rockford, IL, U.S.A. Antibody binding was detected by using the SuperSignal chemiluminescence kit (Pierce) and an Alpha, Innotech imaging system (Santa Clara, California, U.S.A).
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