CK (Figure 1A ) (purity 98%) and 5-fluorouracil(5-Fu) were purchased from Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China). Dulbecco’s modified Eagle’s medium (DMEM) and penicillin-streptomycin were purchased from Gibco-BRL (Grand Island, NY, USA), and fetal bovine serum (FBS) was obtained from BI. Rabbit-polyclonal antibodies against Bclaf1, HIF-1α, LDHA, PHD, and RACK1 were purchased from Abcam (Cambridge, MA, USA). Human-polyclonal antibody against HIF-1α was purchased from Abcam (Cambridge, MA, USA). Rabbit-polyclonal antibodies against Glut1, PDK1, HK2, HSP70, HSP90, and β-actin were purchased from Wanleibio (Shenyang, China). Secondary antibodies were purchased from ZSGB-Bio Co., Ltd. (Beijing, China). The CCK8 assay was obtained from Dojindo (Tokyo, Japan) and the cell cycle analysis kit was from Baihao (Shenyang, China).
Rack1
Manufactured by Abcam
Sourced in United States, United Kingdom, Denmark
RACK1 is a 36 kDa protein that functions as a scaffold, facilitating the assembly of signaling complexes within the cell. It is ubiquitously expressed and plays a key role in various cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Cck 8 assay, by Dojindo Laboratories (1 mentions)
Hif 1α, by Abcam (1 mentions)
5 fluorouracil 5 fu, by Yuanye Bio-Technology (1 mentions)
β actin, by Wanlei (1 mentions)
Mini gel system, by Bio-Rad (1 mentions)
Protease and phosphatase inhibitor, by Roche (1 mentions)
Fluorescent dye labeled secondary antibody, by Thermo Fisher Scientific (1 mentions)
N ethylmaleimide, by Merck Group (1 mentions)
Bcl 2, by Abcam (1 mentions)
Caspase 3, by Abcam (1 mentions)
Bcl2 associated x (bax), by Abcam (1 mentions)
Odyssey infrared imaging system, by LI COR (1 mentions)
Cyt c, by Abcam (1 mentions)
Phosphor akt, by Cell Signaling Technology (1 mentions)
Phospho erk, by Cell Signaling Technology (1 mentions)
Lsab2 system, by Agilent Technologies (1 mentions)
Enhanced chemiluminescence, by Merck Group (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
4 protocols using rack1
1
Molecular Mechanisms in Cancer Metabolism
2
Western Blot Analysis of Apoptosis Regulators
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Epidermis and keratinocytes were lysed in denaturing RIPA lysis buffer supplemented with 20 mM N-ethylmaleimide (Sigma, USA), protease, and phosphatase inhibitors (Roche, Mannheim, Germany). SDS-PAGE (10%) was carried out using the mini-gel system from Bio-Rad. Proteins were transferred to a nitrocellulose membrane. The membrane was blocked with 5% non-fat milk in PBST buffer for 1 h at room temperature, then the membrane was incubated overnight at 4° C with specific primary antibodies against Fam114A1, RACK1, Bax, Bcl2, Caspase 3, cytc, P53, and GADPH/Actin (all 1:1000; Abcam, Cambridge, UK). The membrane was then washed with pBST buffer and incubated with fluorescent dye-labeled secondary antibody (Invitrogen, Carlsbad, CA, USA) for 1 h at room temperature in the dark. The proteins were visualized using an Odyssey Infrared Imaging System (LI-COR, Lincoln, Nebraska, USA).
3
Immunohistochemistry Analysis of Signaling Proteins
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Briefly, slides were dehydrated in xylene and a graded alcohol series. Antigen retrieval was carried out with 0.01 M citrate buffer (pH 6.0) at 95°C for 10 min. Then slides were incubated with diluted primary antibodies against RACK1 (Abcam; 1:40), phospho-ERK (Cell Signaling Tech; 1:50), and phosphor-Akt (Cell Signaling Tech; 1:50) for 12 h, followed by incubations with biotinylated secondary antibody for 1 h, peroxidase-labeled streptavidin for 15 min (LSAB-2 System; DAKO, Glostrup, Denmark), and diaminobenzidine and hydrogen peroxide chromogen substrate plus diaminobenzidine enhancer (DAKO) for 10 min. Slides were counterstained with Mayer's hematoxylin. Known positive and negative control tissues were processed at the same time and under the same conditions.
4
Immunoblotting Analysis of Adiponectin Receptors
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Immunoblotting was conducted as described previously [27 (link), 32 (link)]. The membranes were probed with primary antibodies against adipoR1 (rabbit, 1:1000, Abcam), adipoR2 (rabbit, 1:1000, Abcam), RACK1 (rabbit, 1:1000, Abcam), PKCK2α (rabbit, 1:1000, Cell Signaling Technology), CaMKII (rabbit, 1:1000, Abcam), phospho-CaMKII (mouse, 1:800, Abcam), PKCβ1 (mouse, 1:1000, ThermoFisher Scientific), Cav3.2 (rabbit, 1:800, Alomone) and GAPDH (rabbit, 1:3000, Cell Signaling Technology). Blots were washed and subsequently probed with horseradish peroxidase (HRP)-conjugated goat anti-rabbit or goat anti-mouse IgG secondary antibody (1:5000, Abcam). The immunocomplexes were detected with enhanced chemiluminescence (Merck Millipore). The Chin-X Imager System (Shanghai, China) was used to detect the bands, and NIH ImageJ software was used to quantify the protein band intensities.
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