Hsp40

Manufactured by Cell Signaling Technology

Sourced in United States

Hsp40 is a family of co-chaperone proteins that function in protein folding and transportation processes within cells. These proteins play a crucial role in regulating the activity of Hsp70 chaperones, which are essential for maintaining cellular protein homeostasis.

Automatically generated - may contain errors

Lab products found in correlation

Spelling variants of this product

Anti-HSP40 (4 citations)

10 protocols using hsp40

Platelet Proteome Analysis via Western Blot

Check if the same lab product or an alternative is used in the 5 most similar protocols

After treatment with the indicated inhibitors with or without
thrombin-mediated activation, platelet whole cell lysates were generated in TNES
buffer (50mM Tris pH7.5, 100mM NaCl, 2mM EDTA, 1% NP-40 supplemented with
protease and phosphatase inhibitors (cOmplete and PhosSTOP cocktail tablets,
respectively, Sigma-Aldrich, St. Louis, MO, USA). After cellular debris was
removed via high speed centrifugation, equal amounts of the lysates were
fractionated on gradient 4–20% SDS-PAGE gels and transferred to
nitrocellulose membranes (both from Bio-Rad, Hercules, CA, USA). The membranes
were analyzed for immunoreactivity to the following antibodies: Hsp40, (Cell
Signaling Technology, Danvers, MA, USA), Hsp70, Hsp90, and Grp94 (Enzo Life
Sciences, Farmingdale, NY, USA), and α-tubulin (EMD Millipore,
Burlington, MA, USA). Bound antibodies were detected via species-specific
horseradish peroxidase (HRP)- conjugated secondary antibodies at a 1:000
dilution (GE Healthcare, Pittsburgh, PA, USA), followed by reaction with Pico
ECL (Thermo Scientific, Rockford, IL, USA) to produce a chemiluminescent signal,
and exposed to x-ray film (Vita Scientific, College Park, MD, USA).

Jackson J.W., Rivera-Marquez G.M., Beebe K., Tran A.D., Trepel J.B., Gestwicki J.E., Blagg B.S., Ohkubo S, & Neckers L.M. (2020). Pharmacologic dissection of the overlapping impact of heat shock protein family members on platelet function. Journal of thrombosis and haemostasis : JTH, 18(5), 1197-1209.

+ Open protocol

+ Expand

Western Blot Immunodetection of Molecular Chaperones

Cited 1 time

Check if the same lab product or an alternative is used in the 5 most similar protocols

Western blots were performed as previously described^{16 (link)}. Antibodies (Abs) were used as follows: rabbit anti-HSP90, cHSP70, HSP40, HSP27, HSF1, PSD95, Synapsin I (1:1000; Cell Signaling); mouse anti-iHSP70 (1:1000; Enzo Life Sciences), rabbit anti-BDNF (1:500; Santa Cruz Biotechnology); mouse anti-β-actin (1:10000; Sigma-Aldrich), rabbit anti-Synaptophysin (1:2000; Chemicon/Millipore), and anti-mouse IgG and anti-rabbit IgG horseradish peroxidase-conjugated Abs (1:5000; Sigma-Aldrich).

Wang B., Liu Y., Huang L., Chen J., Li J.J., Wang R., Kim E., Justicia C., Sakata K., Chen H., Planas A., Ostrom R.S., Li W., Yang G., McDonald M.P., Chen R., Heck D, & Liao F.F. (2016). A CNS-permeable Hsp90 inhibitor rescues synaptic dysfunction and memory loss in APP-overexpressing Alzheimer’s mouse model via an HSF1-mediated mechanism. Molecular psychiatry, 22(7), 990-1001.

+ Open protocol

+ Expand

Western Blot Antibody Validation

Check if the same lab product or an alternative is used in the 5 most similar protocols

Antibodies against the following proteins were purchased from the indicated companies: α-Tubulin (Sigma-Aldrich); BAG3 (OriGene); BiP, Caspase-3 (8G10), c-Fos, CHOP, EGR-3, eIF2α, eIF2αx-pS51, FLIP (D16A8), FosB, HSF-1, HSP40, HSP70, IRE1α, PARP, PAR-4, PERK, TAZ, and YAP (Cell Signaling); EGR-1 and Withaferin A (Santa Cruz Biotechnology); GAPDH (Fitzgerald); HSPA6 (Abcam); LC3 (MBL); and vimentin (ARP).

Nishikawa Y., Okuzaki D., Fukushima K., Mukai S., Ohno S., Ozaki Y., Yabuta N, & Nojima H. (2015). Withaferin A Induces Cell Death Selectively in Androgen-Independent Prostate Cancer Cells but Not in Normal Fibroblast Cells. PLoS ONE, 10(7), e0134137.

+ Open protocol

+ Expand

Protein Expression Analysis Post-Transfection

Check if the same lab product or an alternative is used in the 5 most similar protocols

Samples were prepared 72 hrs post-transfection for molecular analyses unless otherwise specified. For the examination of Caspase-3 samples were prepared between 72–96 hrs. In brief, cells were fed every 48 hrs, and samples were collected by centrifugating the media, pelleting the cells. Also, adherent cells were scrapped in PBS, pelleted and combined with the cells in the media. Thereupon, samples were lysed in the presence of non-ionic detergents containing protease inhibitors, and the samples were boiled in the presence of 6X-SDS. Conventional Western blot protocols were used with the following antibodies. Primary Antibodies: G6PDH was purchased from Sigma (A9521). All other antibodies were purchased from Cell Signaling Inc.: HSF1 (No. 4356), Hsp90 (No. 4875), Hsp70 (No. 4872), Hsp60 (No. 4870), Hsp40 (No. 4868), Calnexin (No. 2433), GRP78 (No. 3177), EGFR∼p (No. 2234), Erk1/2∼p (No. 9146), total Erk1/2 (No. 9102), Caspase-3 (#9662). The PARP antibody was anti PARP-DBD, a gift from Dr. W.L. Kraus, and transglulaminase (TGM2) was a gift from Dr. R. Cerione's laboratory purchased from Thermo Scientific. Secondary antibodies were used according to proper immunoreactivity. PVDF membranes were blocked using 5% BSA and membranes were incubated primary antibodies overnight at 4°C.

Salamanca H.H., Antonyak M.A., Cerione R.A., Shi H, & Lis J.T. (2014). Inhibiting Heat Shock Factor 1 in Human Cancer Cells with a Potent RNA Aptamer. PLoS ONE, 9(5), e96330.

+ Open protocol

+ Expand

Immunohistochemical Analysis of Hsp40 Expression

Cited 1 time

Check if the same lab product or an alternative is used in the 5 most similar protocols

In brief, slides were incubated 1 hour in a 60° oven and rehydrated using a gradient of xylene and ethanol. Antigen Retrieval was performed with 10 mM Citrate Buffer. The slides were blocked for 20 minutes, then incubated overnight at 4 °C with primary antibody. Detection was performed as previously described [17 (link), 18 (link), 19 (link)]. For statistical analyses, an average of three fields of view per sample was used for counting and the average percent positive cells were counted and graphed. Statistical analyses were performed using the Student's t-test with p ≤ 0.05 establishing significance. The following antibody was used: Hsp40 (Cell Signaling cat. 4871).

Ignacio D.N., Mason K.D., Hackett-Morton E.C., Albanese C., Ringer L., Wagner W.D., Wang P.C., Carducci M.A., Kachhap S.K., Paller C.J., Mendonca J., Li-Ying Chan L., Lin B., Hartle D.K., Green J.E., Brown C.A, & Hudson T.S. (2019). Muscadine grape skin extract inhibits prostate cancer cells by inducing cell-cycle arrest, and decreasing migration through heat shock protein 40. Heliyon, 5(1), e01128.

+ Open protocol

+ Expand

Comprehensive Protein Analysis of Cell Lysates

Check if the same lab product or an alternative is used in the 5 most similar protocols

Protein analysis of cell lysates was determined with the BioRad DC Protein assay. Twenty to thirty micrograms of protein was fractionated on SDS gels and probed with the indicated antibodies: HSP110 1:1000, (Novus Biologicals), HSP90α HRP conjugate (1:20,000) and β, 1:1000, (Enzo Life Sciences); HSP70 1:5000, HSP27 1:1000, p53 at 1:1000 (from StressMarq), HSP60, HSP40 1:1000 (Cell signaling). Other antibodies used in this study, pErk, pAkt, total Erk and Akt, PI3K (110), pTEN, BRM, TSC1, FOXO, HER2, p21 and MYC all from Cell Signaling (1:1000). Antibodies for cell cycle analysis were from Abcam (cell cycle and apoptosis antibody kit.). Loading controls were GAPDH 1:1000 (Santa Cruz) or actin 1:5000 (Sigma). Secondary antibodies were from Jackson Immunolabs and were used at 1:20,000). Detection was by Super Signal WestPico Chemeluminescence (Thermo Scientific). Images were quantitated with Alpha Ease Imaging Unit using Alpha Ease FC Standalone Software.

Cavanaugh A., Juengst B., Sheridan K., Danella J.F, & Williams H. (2015). Combined inhibition of heat shock proteins 90 and 70 leads to simultaneous degradation of the oncogenic signaling proteins involved in muscle invasive bladder cancer. Oncotarget, 6(37), 39821-39838.

+ Open protocol

+ Expand

Protein Expression Analysis in Tissue

Check if the same lab product or an alternative is used in the 5 most similar protocols

Total protein was extracted from frozen tissue samples by homogenization in RIPA buffer with additional protease and phosphatase inhibitors (Thermo Scientific, Rockford, IL, USA), followed by centrifuged at 14,000 g at 4°C for 15 min and with the resultant supernatant used for all studies. Equal amounts of protein samples were separated by electrophoresis using SDS-PAGE and then transferred to a polyvinylidene fluoride membrane (Millipore, Billerica, MA, USA). Primary antibodies and their sources used in this study: phosphorylated and total ribosomal protein S6, Calnexin, Hsp40, Hsp60, Hsp70, LC3-B, GAPDH (Cell Signaling, Beverly, MA) and PSMB5, PSMB8, Clp protease (ClpP) (Abcam, Cambridge, MA). Alkaline phosphatase-conjugated secondary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) followed by ECL reagent were used to visualize protein bands which were using ImageJ. When possible, protein samples from all subjects involved in study were analyzed on a single immunoblot for individual protein tested. For LC3B assessment, data were collected over multiple immunoblots; for each immunoblot, both control and rapamycin-treated samples were analyzed concurrently. For all, data were normalized to relative Ponceau S staining of membrane following all antibody visualization.

Lelegren M., Liu Y., Ross C., Tardif S, & Salmon A.B. (2016). Pharmaceutical inhibition of mTOR in the common marmoset: effect of rapamycin on regulators of proteostasis in a non-human primate. Pathobiology of Aging & Age Related Diseases, 6, 10.3402/pba.v6.31793.

+ Open protocol

+ Expand

Hsp Proteins and Resveratrol Study

Check if the same lab product or an alternative is used in the 5 most similar protocols

Antibodies used in this study were as follows: Hsp27, Hsp40, Hsp70, Hsp90, NFϰ-B/p65, and Cyclin D1 (Cell Signaling), and VEGF (Santa Cruz), and actin (Chemicon). Plasmids used in this study were Hsp40 CRISP/Cas9 KO (Santa Cruz), and Control CRISP/Cas9 (Santa Cruz). Resveratrol was purchased from the Sigma Chemical Company (St. Louis, MO).

+ Open protocol

+ Expand

Western Blot Immunodetection of Molecular Chaperones

Cited 1 time

Check if the same lab product or an alternative is used in the 5 most similar protocols

+ Open protocol

+ Expand

Unfolded Protein Response Pathway Assay

Check if the same lab product or an alternative is used in the 5 most similar protocols

Maneb (Product number 45554) and tunicamycin (Product number T7765) were purchased from Sigma-Aldrich. 4μ8c (Product number 412512) was purchased from EMD Millipore. All other reagents were purchased from Fisher-Scientific. The following primary antibodies were purchased from Cell Signaling Technology: XBP1s (D2C1F) (12782), ubiquitin (3933), phospho-eIF2α (Ser51) (9721), eIF2α (9722), IRE1α (3294), HSP40 (4871), HSP70 (4872), HSP90 (4877). The following primary antibodies were purchased from Abcam: ATF6 (ab122897), and XBP1 (ab37152). The primary antibody against phospho-IRE1α (Ser724) was purchased from Novus Biologicals. The primary antibody against HSP27 was purchased by Enzo Life Sciences (G3.1). The primary antibody against β-actin (A5441) was purchased from Sigma-Aldrich.

Aivazidis S., Coughlan C.M., Rauniyar A.K., Jiang H., Liggett L.A., Maclean K.N, & Roede J.R. (2017). The burden of trisomy 21 disrupts the proteostasis network in Down syndrome. PLoS ONE, 12(4), e0176307.

+ Open protocol

+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!