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Hsc70 b6

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HSC70 (B6) is a recombinant protein that represents the heat shock 70 kDa protein 8 (HSPA8), a member of the heat shock protein 70 family. This protein functions as a molecular chaperone, assisting in the folding and unfolding of other proteins.

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Lab products found in correlation

α tubulin, by Cell Signaling Technology (2 mentions) Pvdf membrane, by Bio-Rad (2 mentions) Phosstop, by Roche (2 mentions) Angptl2, by R&D Systems (2 mentions) Ecl kit, by GE Healthcare (2 mentions) Supersep ace gel, by Fujifilm (2 mentions) Sumo1, by Cell Signaling Technology (1 mentions) Antibodies against p53, by Santa Cruz Biotechnology (1 mentions) Westar supernova, by Cyanagen (1 mentions) Animage quant las4000, by GE Healthcare (1 mentions) Tsg101 c 2, by Santa Cruz Biotechnology (1 mentions) Cd81 h 121, by Santa Cruz Biotechnology (1 mentions) Rabbit anti mouse igg, by GeneTex (1 mentions) Goat anti rabbit igg, by GeneTex (1 mentions) P21 waf1 cip1 12d1, by Cell Signaling Technology (1 mentions) Nitrocellulose membrane, by Cytiva (1 mentions) Total stat3, by Santa Cruz Biotechnology (1 mentions) Supersignal west femto maximum sensitivity substrate, by Thermo Fisher Scientific (1 mentions) Complete mini protease inhibitor cocktail, by Roche (1 mentions) β actin, by Abcam (1 mentions)

11 protocols using hsc70 b6

1

Western Blot Analysis of Cellular Proteins

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Total cell lysates and cytosolic and nuclear extracts were prepared as described [31 (link)]. Samples were electrophoresed through 10% SDS–PAGE, followed by transfer to nitrocellulose membranes. Blots were blocked with 5% skim milk and incubated overnight with antibodies listed below. Antibodies against IGF1R β-subunit (#3027), insulin receptor β-subunit (#3025), phospho-p53 (#9284) and SUMO-1 (#5718) were from Cell Signaling Technology (Danvers, MA, USA). Antibodies against p53 (mixture: DO-1 and 1801) and heat shock cognate HSC70 (#B-6) were from Santa Cruz Biotechnology (Dallas, TX, USA). An antibody against lamin B was from Abcam (Cambridge, UK). Blots were washed and incubated with the appropriate horseradish peroxidase-conjugated secondary antibody. Protein A-peroxidase conjugated was used for p53 detection (Rockland Immunochemicals Inc, Limerick, PA, USA). Proteins were detected using the enhanced chemiluminiscence reaction (Westar Supernova, Cyanagen, Bologna, Italy). HSC70 was used as a loading control and lamin B as a nuclear marker.
Sarfstein R, & Werner H. (2020). Tumor suppressor p53 regulates insulin receptor (INSR) gene expression via direct binding to the INSR promoter. Oncotarget, 11(25), 2424-2437.
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2

Isolation and Analysis of Extracellular Vesicles

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To prepare brain homogenates, tissue was removed from the frontal lobe,
lysed in strong lysis buffer (detailed above) with proteinase inhibitor,
homogenized by pipette, and centrifuged at 10,000 × g for 10 minutes to
discard whole cells and intact cellular debris. For confirmation of EV proteins
in purified lysates, 5X Laemmli sample buffer [10% SDS, 250 mM Tris pH 6.8, 1
mg/mL bromophenol blue, 0.5 M DTT, 50% glycerol, 5% BME] was added to EV
isolates for a final concentration of 1X. For immunoblot analysis of CD63, DTT
and BME were omitted from lysis and sample buffers to allow the protein to run
in non-reducing conditions. Gel electrophoresis and western blotting was
performed as previously described (Hurwitz et
al., 2017b). Equal volume of gradient lysates were loaded into an
SDS-PAGE gel. Blots were probed with the following antibodies: Alix (Q-19; Santa
Cruz Biotechnology), HSC70 (B-6; Santa Cruz), TSG101 (C-2; Santa Cruz), CD63
(TS63; Abcam), Rab8a (63-BJ ; Santa Cruz), and CD81 (H-121 ;Santa Cruz), rabbit
anti-mouse IgG (Genetex, 26728), rabbit anti-goat IgG (Genetex, 26741), goat
anti-rabbit IgG (Fab fragment) (Genetex, 27171). Blots were imaged using an
Image Quant LAS4000 (General Electric) and processed with ImageQuant TL v8.1.0.0
software, Adobe Photoshop CS6 and CorelDraw Graphic Suite X5.
Hurwitz S.N., Sun L., Cole K.Y., Ford CR I.I.I., Olcese J.M, & Meckes DG J.r. (2018). An optimized method for purification of whole brain-derived extracellular vesicles reveals insight into neurodegenerative processes in a mouse model of Alzheimer’s disease.. Journal of neuroscience methods, 307, 210-220.
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3

Comprehensive Protein Extraction and Western Blot Analysis

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Cell lysates were prepared with RIPA buffer containing 20 mM Tris-HCl pH 7.5, 10 mM EDTA, 1% sodium deoxycholate, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, phosphatase inhibitor consisting of 2 mM imidazol, 1 mM sodium orthovanadate and 1 mM sodium fluoride, and cOmplete™ mini protease inhibitor cocktail (Roche). Samples were lysed in RIPA buffer with sonication. Protein concentrations were determined by BCA protein assay (Pierce). Equal amounts of lysates were loaded (15–30 µg) and separated by SDS-polyacrylamide gel electrophoresis followed by transfer onto nitrocellulose membranes (Amersham). After blocking with 5% milk (Roth), membranes were incubated with the following antibodies: HSC70 [B-6] (Santa Cruz), beta-Actin (Abcam), total-AKT [D9E] (Cell Signaling), p53 [DO-1] or HRP-conjugated p53 [DO-1] (Santa Cruz), phospho-Y705 STAT3 [EP2147Y] (Abcam), total STAT3 (Santa Cruz) or total STAT3 [79D7] (Cell Signaling), MDM2 [IF-2] (Calbiochem®/Millipore), p21 Waf1/Cip1 [12D1] (Cell Signaling). Primary antibodies were detected with HRP-conjugated secondary antibodies. Signal was developed using Clarity Max™ Western ECL Substrate (BioRad), SuperSignal™ West Femto Maximum Sensitivity Substrate (ThermoFisher Scientific), or Immobilion Western chemiluminescent HRP substrate (Millipore/Merck). For antibody details, see Supplementary Table 1.
Klemke L., Fehlau C.F., Winkler N., Toboll F., Singh S.K., Moll U.M, & Schulz-Heddergott R. (2021). The Gain-of-Function p53 R248W Mutant Promotes Migration by STAT3 Deregulation in Human Pancreatic Cancer Cells. Frontiers in Oncology, 11, 642603.
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4

Western Blot Analysis of Proteins

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Protein lysates were heated with LDS buffer and reducing agent (Life sciences) before running on an SDS-PAGE gel and transferred to a PVDF membrane (Bio-Rad). Membranes were blocked using 5% BSA or Odyssey blocking buffer (PBS) and then incubated with the following target-specific antibodies: LARS (Cell Signaling, 13868S, 1;1000), HSC70 (B6) (Santa Cruz, sc-7298, 1:3000), PyMT (Novus Biologicals, NB100–2749, 1:2500), EMP3 (Abcam, ab236671, 1:500), GGT5 (GeneTex, GTX81477, 1:500), α-tubulin (Cell Signaling, 3873S, 1:1000), QARS (Proteintech, 12645–1-AP, 1:1000), HARS (Proteintech, 16375–1-AP, 1:1000), NARS (Aviva Systems Biology, OAAB07325, 1:1000), IARS (ThermoFisher, PA5–44246, 1:1000), HA (BioLegend, 901501, 1:1000), Luciferase (Proteintech, 27986–1-AP, 1:1000).
Passarelli M.C., Pinzaru A.M., Asgharian H., Liberti M.V., Heissel S., Molina H., Goodarzi H, & Tavazoie S.F. (2022). Leucyl-tRNA synthetase is a tumour suppressor in breast cancer and regulates codon-dependent translation dynamics. Nature cell biology, 24(3), 307-315.
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5

Whole Cell Lysate Preparation and Western Blotting

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Whole cell lysates (WCL) were prepared by lysing cells in lysis buffer [50 mM Tris pH 7.6, 150 mM NaCl, 1% Triton X-100, 0.5% deoxycholate, 0.1% SDS, 1 mM sodium ortho-vanadate, 5 mM sodium fluoride, 50 µg/mL phenylmethylsulfonyl fluoride (PMSF), protease inhibitor cocktail mix 1 (Calbiochem) and PhosSTOP (Roche)]. WCL were resolved by SDS-PAGE on 4–12% gradient polyacrylamide gels (Invitrogen) at 180 V for 1 hr and transferred electrophoretically by wet transfer system onto PVDF membrane (Milipore) at 100 V for 1 hr. Blots were blocked for 1 hr with 5% skimmed milk, rinsed and probed with the required primary antibody (diluted in 5% BSA, 0.1% Tween-20 containing TBS and 0.05% sodium azide) overnight at 4 °C. Following incubation with appropriate horseradish peroxidase-conjugated secondary antibody (Cell Signalling, 7076 or 7074, 1:5000) (in 5% skimmed milk), bands were visualised using Bio-RAD ChemiDoc™ Imaging System and software. Antibodies used were: ERK5 (Cell Signalling, 3372, 1:1000) and HSC70 (B-6) (Santa Cruz Biotechnology, sc-7298, 1:1000). Densitometry was performed using Image J software.
Loveridge C.J., van ’t Hof R.J., Charlesworth G., King A., Tan E.H., Rose L., Daroszewska A., Prior A., Ahmad I., Welsh M., Mui E.J., Ford C., Salji M., Sansom O., Blyth K, & Leung H.Y. (2017). Analysis of Nkx3.1:Cre-driven Erk5 deletion reveals a profound spinal deformity which is linked to increased osteoclast activity. Scientific Reports, 7, 13241.
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6

Immunoblot Analysis of Cell Signaling Proteins

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Immunoblot analysis was performed as described24 (link). SuperSep Ace gels (5–20%) were from Wako. Antibodies were against ETS1 (Abcam, Cambridge, UK), HSC70 (B-6, Santa Cruz Biotechnology), CXCR4 (ab124824, ab2074; Abcam), and ANGPTL2 (R&D Systems). Immunodetection was performed using an enhanced chemiluminescence (ECL) kit (GE Healthcare, Amersham, UK).
Masuda T., Endo M., Yamamoto Y., Odagiri H., Kadomatsu T., Nakamura T., Tanoue H., Ito H., Yugami M., Miyata K., Morinaga J., Horiguchi H., Motokawa I., Terada K., Morioka M.S., Manabe I., Iwase H., Mizuta H, & Oike Y. (2015). ANGPTL2 increases bone metastasis of breast cancer cells through enhancing CXCR4 signaling. Scientific Reports, 5, 9170.
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7

Western Blot Analysis of Proteins

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Protein lysates were heated with LDS buffer and reducing agent (Life sciences) before running on an SDS-PAGE gel and transferred to a PVDF membrane (Bio-Rad). Membranes were blocked using 5% BSA or Odyssey blocking buffer (PBS) and then incubated with the following target-specific antibodies: LARS (Cell Signaling, 13868S, 1;1000), HSC70 (B6) (Santa Cruz, sc-7298, 1:3000), PyMT (Novus Biologicals, NB100–2749, 1:2500), EMP3 (Abcam, ab236671, 1:500), GGT5 (GeneTex, GTX81477, 1:500), α-tubulin (Cell Signaling, 3873S, 1:1000), QARS (Proteintech, 12645–1-AP, 1:1000), HARS (Proteintech, 16375–1-AP, 1:1000), NARS (Aviva Systems Biology, OAAB07325, 1:1000), IARS (ThermoFisher, PA5–44246, 1:1000), HA (BioLegend, 901501, 1:1000), Luciferase (Proteintech, 27986–1-AP, 1:1000).
Passarelli M.C., Pinzaru A.M., Asgharian H., Liberti M.V., Heissel S., Molina H., Goodarzi H, & Tavazoie S.F. (2022). Leucyl-tRNA synthetase is a tumour suppressor in breast cancer and regulates codon-dependent translation dynamics. Nature cell biology, 24(3), 307-315.
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8

Immunoprecipitation and Western Blot Analysis

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Cell lysis, immunoprecipitation, blotting and blocking was performed as
described (55 (link)). For western blot analysis
of initially transformed cells, density gradient centrifugation
(Histopaque®-1077, Sigma-Aldrich) was performed to isolate viable
lymphocytes. NFYA was immunoprecipitated using the NFYA (G-2, Santa Cruz
Biotechnology) antibody. For immunoprecipitation of HA-tagged CDK6,
Pierce™ Anti-HA Magnetic Beads (Thermo Fisher Scientific) were used. The
following antibodies were used for detection: PRMT5 (07-405, Merck-Millipore),
GAPDH (ABS16, Merck-Millipore), pY207Crkl (Cell Signaling
Technologies), pS780Rb (Cell Signaling Technologies), HA (ab9110,
Abcam), CDK4 (H-22), CDK6 (H96 and DCS90), NFYA (G-2), β-ACTIN (AC-15),
BCL2 (N-19), p53 (DO-1), p21 (F-5) and HSC70 (B-6) all from Santa Cruz
Biotechnology.
Bellutti F., Tigan A.S., Nebenfuehr S., Dolezal M., Zojer M., Grausenburger R., Hartenberger S., Kollmann S., Doma E., Prchal-Murphy M., Uras I.Z., Höllein A., Neuberg D.S., Ebert B.L., Ringler A., Mueller A.C., Loizou J.I., Hinds P.W., Vogl C., Heller G., Kubicek S., Zuber J., Malumbres M., Farlik M., Villunger A., Kollmann K, & Sexl V. (2018). CDK6 antagonizes p53-induced responses during tumorigenesis. Cancer discovery, 8(7), 884-897.
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9

Protein Immunoblotting Characterization

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Protein extracts were subjected to SDS‐PAGE, and proteins were electrotransferred to PVDF membrane. Immunoblotting was undertaken with Abs against TFE3 (Sigma‐Aldrich), GAPDH (D16H11; Cell Signaling Technology, Danvers, MA, USA), histone H3 (D1H2; Cell Signaling Technology), and Hsc70 (B‐6; Santa Cruz Biotechnology, Dallas, TX, USA). Detailed information relevant to protein extraction from cultured cells is provided in Data S1.
Kurahashi R., Kadomatsu T., Baba M., Hara C., Itoh H., Miyata K., Endo M., Morinaga J., Terada K., Araki K., Eto M., Schmidt L.S., Kamba T., Linehan W.M, & Oike Y. (2019). MicroRNA‐204‐5p: A novel candidate urinary biomarker of Xp11.2 translocation renal cell carcinoma. Cancer Science, 110(6), 1897-1908.
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10

Worm Protein Extraction and Western Blotting

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Protein sample preparations were performed by collecting worms from three full plates and whasing them at least three times in M9 buffer. Worm pellet was frozen in liquid nitrogen and, depending on the amount of worm pellet, liquefied in 100–200 μM worm lysis buffer (25 mM Tris-HCL pH 7.4, 0.15 M NaCl, 1 mM EDTA, 1% NP-40, 0.5% SDS, 10 mM DTT and proteinase inhibitor cocktail (Roche)). PhosStop (Roche) was added when phospho-antibody was used. Samples were sonicated using the BioruptorR Sonication device (Diagenode) six times at 30/60 cycles. Samples were then centrifugaed for 15 min at 16,000×g at 4 °C and the supernatant was used. Protein concentration was measured with Bradford assay. Western blotting was performed using antibodies against ATP-1 (ATP5A, 1:2000 Abcam, #ab14748), GFP (1:2000, OriGene, #TP401), HSP-1 (HSC70 (B6), 1:4000, Santa Cruz, #sc-7298). LONP-1 (1:2000 Proteintech, #15440-1-AP), NUO-1 (NDUFV1, 1:2000 Proteintech, #11238-1-AP), NUO-2 (NDUFS3, 1:2000 MitoSciences, #MS112), Phospho-p38 MAPK (1:2000, Cell Signaling, #4511) and Phospho-(Ser/Thr) Phe (1:2000, Cell Signaling, #9631). Data were normalized to HSP-1.
Hermeling J.C., Herholz M., Baumann L., Cores E.C., Zečić A., Hoppe T., Riemer J, & Trifunovic A. (2022). Mitochondria-originated redox signalling regulates KLF-1 to promote longevity in Caenorhabditis elegans. Redox Biology, 58, 102533.
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