Harvested cell pellets were lysed in (100–200 μl) of RIPA buffer (Sigma-Aldrich) and then proteins were extracted and quantified. 10 µg of extracted lysate was resolved in SDS-polyacrylamide gel electrophoresis (SDS-PAGE), followed by electroblotting onto methanol-activated PVDF membranes (Millipore, USA) using a semidry transfer unit (ATTO, Japan). Immunoblotting was performed with antibodies against caspase-9 (sc-7885), caspase-3 (sc-7148), Cyclin D1 (sc-450), Cyclin A (sc-239), Vimentin (sc-6260), β-catenin (sc-7963), CDK4 (sc-260), CDK2 (sc-163), PARP 1/2 (sc-7150), ATR (SC-28901), pATR (sc-109912), purchased from Santa Cruz. hnRNP-K (#4675), MMP-9 (#2270), p21WAF1 (#2947), SMAD-2/3 (#8685) and CHK-1 (#2345) were procured from Cell Signaling Technologies. Antibody for CARF (rabbit polyclonal) was generated endogenously in the laboratory. The immunoblots were incubated with horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit antibodies (Santa Cruz Biotechnology) and detected using ECL substrate (GE Healthcare, NJ, USA). Densitometric quantitation of the representative immunoblots was carried out using the ImageJ software from NIH (National Institute of Health). All the experiments were performed in triplicate.
Hnrnp k
Manufactured by Cell Signaling Technology
Sourced in United States
HnRNP-K is a nuclear protein that plays a role in the packaging and processing of mRNA. It is involved in the regulation of transcription, translation, and mRNA stability.
Automatically generated - may contain errors
Lab products found in correlation
Mmp 2, by Santa Cruz Biotechnology (2 mentions)
Vascular endothelial growth factor (vegf), by Santa Cruz Biotechnology (2 mentions)
Axiovert 200m, by Zeiss (2 mentions)
Ecl substrate, by GE Healthcare (1 mentions)
Smad2 3, by Cell Signaling Technology (1 mentions)
Vimentin sc 6260, by Santa Cruz Biotechnology (1 mentions)
P21waf1, by Cell Signaling Technology (1 mentions)
Methanol activated pvdf membrane, by Merck Group (1 mentions)
Mmp 9, by Cell Signaling Technology (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Fibronectin, by Santa Cruz Biotechnology (1 mentions)
Vimentin, by Santa Cruz Biotechnology (1 mentions)
Mda mb 231, by Japanese Collection of Research Bioresources Cell Bank (1 mentions)
Hsc 3, by Japanese Collection of Research Bioresources Cell Bank (1 mentions)
Dld 1, by Japanese Collection of Research Bioresources Cell Bank (1 mentions)
Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (1 mentions)
Mmp 9, by Abcam (1 mentions)
β catenin, by Santa Cruz Biotechnology (1 mentions)
E cadherin, by Cell Signaling Technology (1 mentions)
Collagen 4, by Abcam (1 mentions)
4 protocols using hnrnp k
1
Immunoblotting Analysis of Apoptosis and Cell Cycle Markers
2
Comprehensive Cancer Cell Line Protocol
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Panc-1 (pancreatic adenocarcinoma), MDA-MB-231 (breast adenocarcinoma), HeLa (cervical adenocarcinoma), DLD-1 (colorectal adenocarcinoma), T.T. (esophageal squamous cell carcinoma) and HSC3 (oral squamous cell carcinoma) cells were obtained from the Japanese Collection of Research Bioresources (JCRB), Japan and cultured in Dulbecco’s modified Eagle’s medium (Invitrogen) supplemented with 5% fetal bovine serum and 1% penicillin/streptomycin in a humidified incubator (37 °C and 5% CO2). TEG derivatives TD-10 or triethylene glycol dimethacrylate (261548-250ML) and TD-11 or tetraethylene glycol dimethacrylate (86680-100ML) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Primary antibodies against VEGF (Santa Cruz, Dallas, TX, USA, SC-507), β-actin (AbCam, Cambridge, UK, ab49900), MMP-2 (Santa Cruz, Dallas, TX, USA, SC-13594), MMP-7 (Santa Cruz, Dallas, TX, USA, SC-30071), MMP-9 (AbCam, Cambridge, UK, ab38898), Mortalin [71 (link)], E-cadherin (Cell Signaling, Denvers, MA, USA, 5296S), Collagen IV (AbCam, Cambridge, UK, ab6586), Fibronectin (Santa Cruz, Dallas, TX, USA, SC-52331), Vimentin (Santa Cruz, Dallas, TX, USA, SC-6260), hnRNP-K (Cell Signaling, Denvers, MA, USA, 4675S) and β-catenin (Santa Cruz, Dallas, TX, USA, SC-7963) were used in immunostaining and Western blotting.
3
Immunofluorescence Imaging of Cellular Proteins
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For immunofluorescence study, cells were cultured on coverslips placed in 12-well dish. After overnight incubation, cells were treated with embelin for 24 h, washed with cold PBS and fixed with methanol: acetone (1:1) for 5 min. Fixed cells were washed twice with 1 X PBS, permeabilized using 0.5% Triton X-100 in PBS for 10 min, and blocked using 2% BSA in PBS for 15 min. Coverslips containing cells were incubated with antibodies against TACE, MMP-9, MMP-2, VEGF (Santa Cruz Biotechnology Inc., Texas), hnRNP-K (Cell Signaling Technology Inc., MA) proteins for 2 h at room temperature, washed thrice with 0.2% Triton X-100 in PBS followed by incubation with Alexa Fluor conjugated secondary antibodies. After further washings with 0.2% Triton X-100 in PBS, cells on coverslips were mounted and visualized under Carl Zeiss microscope (Axiovert 200 M).
4
Western Blot Analysis of Protein Targets
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Protease inhibitors blended with RIPA buffer (prepared using sodium deoxycholate [0.5%], Tris [pH 8.0] at 50 mM, SDS [0.1%], sodium chloride at 150 mM, and NP-40 [1%]) were utilized for sample lysis on ice. Coomassie brilliant blue dye method was adopted to examine the protein concentration. Specifically, each lane with equivalent content of proteins was treated by SDS–PAGE gels (10–15%) before nitrocellulose membrane (Millipore) transfer by means of submerged transfer. Following 1 h of room-temperature sealing with 5% fat-free milk or 3% BSA in TBST, diversified primary antibodies were employed to overnight incubate the membrane at 4 °C. As instructed by the manufacturer, an enhanced chemiluminescence Western blotting kit (K-12045-D50; Apgbio, Beijing, China) was employed for signal visualization subsequent to incubation (1 h, room temperature) with secondary antibodies coupled with peroxidase. The development of blots was detected on the Molecular Imager (Bio-Rad, USA). The following antibodies information was presented: AURKA (Upstate, 07–648), HRP-coupled Goat anti-Rabbit IgG (Thermo-Pierce, 31460), AURKA (Sigma, a1231), P-AURKA (Cell Signaling Technology, D13A11), HRP-labeled Goat anti-Mouse IgG (Thermo-Pierce, 31430), GAPDH (KANGCHEN, KC-5G4), hnRNPK (Cell Signaling Technology, 4675S), YBX1 (Santa Cruz Biotechnology, 398340).
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